JP2023106392A - Cd3 antigen binding fragment and application thereof - Google Patents

Abstract

To provide a humanized and multifunctional anti-CD3 antibody which has improved biological activity, thermal stability, and/or acid resistance.SOLUTION: The present invention relates to a humanized antibody or an antigen binding fragment thereof, the antibody specifically binding to CD3 of primates, e.g., humans and/or monkeys, the antibody comprising framework regions FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3 and FR-L4 and complementarity-determining regions (CDRs), wherein the amino acid sequences of CDR1 and CDR2 of heavy chain variable regions, the amino acid sequences of CDR3 of heavy chain variable regions, the amino acid sequences of CDR1, CDR2 and CDR3 of light chain variable regions, and the framework regions of the humanized antibody comprise specific sequences, respectively.SELECTED DRAWING: None

Description

The present invention relates to the field of genetic engineering, particularly to the field of antibody engineering. Specifically, the present invention relates to antibodies, such as multifunctional antibodies, such as antibodies directed against CD3, particularly humanized antibodies, antigen-binding fragments of said antibodies and related uses.

CD3 is a T-cell surface molecule that can bind to T-cell receptors on the surface of T-cells to form a TCR-CD3 complex and activate T-cells. It plays an important role in antigen recognition and immune signal transduction. Anti-CD3 antibodies are widely used for treatment of transplant rejection, autoimmune diseases, and the like. Mouse-derived anti-CD3 antibodies such as OKT3 are known to be unfavorable for use in humans, as they may induce a HAMA (human anti-mouse antibody) reaction. Therefore, there is a need to humanize or otherwise process these murine-derived antibodies to reduce side effects. Traditionally, the main route to avoid or reduce HAMA reactions is to humanize monoclonal antibodies of murine origin or to develop fully humanized antibodies. For example, introducing a sequence fragment similar to a human antibody protein into a murine-derived antibody can reduce the HAMA reaction. However, such treatment may not be possible, as structurally similar proteins may not exist in humans. In addition, antibody humanization is also fraught with technical difficulties, such as reduced antibody affinity, reduced potency, reduced stability or reduced yield, often resulting in no effective therapeutic protein.

WO2013/158856 WO2007/019620 WO2017/023761

The present invention provides antibodies, such as multifunctional antibodies, such as antibodies against CD3, particularly humanized antibodies, antigen-binding fragments of said antibodies and related uses.

In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof, particularly a humanized antibody or antigen-binding fragment thereof, comprising:
specifically binds to CD3 of humans and/or primates such as monkeys;
comprising framework regions FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3 and FR-L4, and complementarity determining regions (CDRs);
The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NOS: 1, 2, and 3, respectively, or variant sequences thereof, such as the variant sequence of CDR-H3 is any one of the sequences shown in SEQ ID NOS: 4-14 and 190-191,
the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28, respectively, or variant sequences thereof;
The framework region of the humanized antibody is
a) FR-H1 of SEQ ID NO: 15 or 16
b) FR-H2 of SEQ ID NO: 17
c) FR-H3 of any one of SEQ ID NOs: 18-24
d) FR-H4 of SEQ ID NO:25
e) FR-L1 of any one of SEQ ID NOs:29-31
f) FR-L2 of any one of SEQ ID NOs: 32-38
g) FR-L3 of any one of SEQ ID NOs:39-42, and/or h) FR-L4 of any one of SEQ ID NOs:43-44
An antibody or antigen-binding fragment thereof comprising one or more sequences selected from the group of

In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof, particularly a humanized antibody or antigen-binding fragment thereof, comprising:
specifically binds to CD3 of humans and/or primates such as monkeys;
comprising a heavy chain variable region and a light chain variable region,
The heavy chain variable region is
a) the amino acid sequence of SEQ ID NO: 45-62,
b) 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or and c) one or more (preferably one or several, more preferably one, two or three ) different amino acids,
wherein the light chain variable region comprises any one sequence selected from the group of
d) the amino acid sequence of SEQ ID NO: 63-73,
e) 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or and f) one or more (preferably one or several, more preferably one, two or three ), an antibody or an antigen-binding fragment thereof comprising any one sequence selected from the group of amino acid sequences having different amino acids from ).

In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof, particularly a humanized antibody or antigen-binding fragment thereof, comprising:
specifically binds to CD3 of humans and/or primates such as monkeys;
comprising a heavy chain variable region and a light chain variable region,
The heavy chain variable region and the light chain variable region, respectively,
a) SEQ ID NOS: 46, 63; SEQ ID NOS: 47, 63; SEQ ID NOS: 49, 63; SEQ ID NOS: 50, 63; SEQ ID NOS: 51, 71; SEQ ID NOS: 52, 72; SEQ ID NOS: 53, 72; SEQ ID NOS: 54, 72; 52, 72; 52, 73; 53, 73; 54, 73; 55, 73; SEQ ID NOs: 45, 63; SEQ ID NOs: 48, 63; SEQ ID NOs: 45, 64; SEQ ID NOs: 45, 67; SEQ ID NOS: 48, 71; SEQ ID NOS: 50, 71; SEQ ID NOS: 61, 72; SEQ ID NOS: 60, 73; SEQ ID NOS: 60, 72;
b) at least one amino acid sequence of a) and 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and c) one or more (preferably one or several, more preferably one, two or three) different amino acids from at least one amino acid sequence of a) An antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group of amino acid sequences having

In some embodiments, the present invention provides: a multispecific antibody, preferably a bispecific antibody, comprising
comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and an antibody or antigen-binding fragment against another antigen and / or other antigenic epitope,
proteins whose other antigens and/or other antigenic epitopes are e.g. overexpressed on tumor cells compared to corresponding non-tumor cells; tumor antigens e.g. CD38, BCMA, PD-L1, SLAMF7, Claudin 18.2 or CEA; viruses; bacteria; and/or multispecific antibodies that are endotoxins.

In some embodiments, the present invention provides: a polypeptide, wherein the amino acid sequence of said polypeptide is any one of SEQ ID NOS:45-62; 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with at least one amino acid sequence or an amino acid sequence having at least one amino acid sequence of SEQ ID NO: 45-62 and one or more (preferably one or several, more preferably one, two or three) different amino acids A polypeptide comprising or selected from these amino acid sequences.

In some embodiments, the present invention provides: a polypeptide, wherein the amino acid sequence of said polypeptide is any one of SEQ ID NOS: 63-73; 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with at least one amino acid sequence or an amino acid sequence having one or more (preferably one or several, more preferably one, two or three) different amino acids from at least one amino acid sequence of SEQ ID NOS: 63-73 A polypeptide comprising or selected from these amino acid sequences.

In some embodiments, the humanized antibody has comparable affinity, improved biological activity, thermal stability and/or acid resistance compared to a control antibody.

In some embodiments, the invention provides polynucleotides encoding the polypeptides of the invention.

In some embodiments, the present invention provides: an antibody comprising (a) a light-heavy chain pair and (b) a fusion peptide, wherein said light-heavy chain pair is associated with a tumor An antibody having specificity for cells or microorganisms, said fusion peptide comprising a single chain variable region fragment and a single chain Fc fragment, and having specificity for immune cells. In some embodiments, said single chain Fc fragment comprises CH2 and/or CH3 as described herein, e.g., CH2 having the sequence of any one of SEQ ID NOs: 155-161 or 192, and/or CH3 having the sequence of any one of SEQ ID NOs: 162-183. In some embodiments, the fusion peptide comprises the corresponding sequence of an antibody described herein or a subsequence thereof. For example, the fusion peptide comprises the sequences of the light and/or heavy chain variable and/or framework regions of the humanized antibodies described herein. In some embodiments, the single chain variable region fragment (scFv) of said fusion peptide comprises the scFv of a humanized antibody described herein.

In some embodiments, the fusion peptide in the antibody of the invention comprises VHs-linker1-VLs-hinge1-CH2-CH3-b and the heavy chain is VHm-CH1-hinge2-CH2-CH3-a and the light chain comprises VLm-CL.

In some embodiments, the light-heavy chain pair in the antibody of the invention is a) a protein that is overexpressed on tumor cells compared to corresponding non-tumor cells, b) a tumor antigen, such as CD38, Binds specifically to BCMA, PD-L1, SLAMF7, Claudin 18.2 or CEA, c) viruses, d) bacteria, and/or e) endotoxins.

In some embodiments, the fusion peptides in the antibodies of the invention specifically bind immune cell antigens. For example, the fusion peptide comprises an antigen binding site that specifically binds to human and/or primate, such as monkey, CD3. For example, the fusion peptide comprises the variable regions of the light and heavy chains of the antibodies described herein.

In some embodiments, the VH of the fusion peptide in the antibody of the invention comprises the sequence of any one of SEQ ID NOS: 45-62, 74, 76, 78, 80, 82, 84, 86, 88, and said fusion VL of the peptide comprises the sequence of any one of SEQ ID NOs: 63-73, 75, 77, 79, 81, 83, 85, 87, 89, and Linker 1 of said fusion peptide comprises any one of SEQ ID NOs: 120-138 wherein hinge 1 of the fusion peptide and hinge 2 of the heavy chain comprise the sequence of any one of SEQ ID NOS: 139-147, and CH2 of the fusion peptide and CH2 of the heavy chain comprise the sequence 155-161 or 192, wherein CH3-b of said fusion peptide is any of SEQ ID NOS: 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183 comprising a sequence, wherein CH3-a of said heavy chain comprises the sequence of any one of SEQ ID NOS: 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182; VHm comprises the sequence of any one of SEQ ID NOS: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 193, and CH1 of said heavy chain comprises the sequence of SEQ ID NO: 154, and the VLm of said light chain is of SEQ ID NOs: and/or the CL of said light chain comprises the sequence of any one of SEQ ID NOS: 148-153.

In some embodiments, the VH and VL of the fusion peptide in the antibody of the invention each comprise an amino acid sequence selected from the group below,
48, 71; 49, 63; 49, 71; 51, 71; 58, 72; SEQ ID NOS: 60, 73; SEQ ID NOS: 59, 72; SEQ ID NOS: 61, 73; SEQ ID NOS: 62, 73; 80, 81; 82, 83; 84, 85; 86, 87; 88, 89; b) at least one amino acid sequence of a) and 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, and c) with at least one amino acid sequence of a) An amino acid sequence having one or more (preferably one or several, more preferably one, two or three) different amino acids, and/or the VHm of the heavy chain and the VLm of the light chain are each: Containing amino acid sequences selected from the group of:
d) SEQ ID NOs: 90, 91; SEQ ID NOs: 92, 93; SEQ ID NOs: 94, 95; SEQ ID NOs: 96, 97; 108, 109; 110, 111; 112, 113; 114, 115; 116, 117;
e) at least one amino acid sequence of d) and 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and f) one or more (preferably one or several, more preferably one, two or three) different amino acids from at least one amino acid sequence of d) Amino acid sequence having

In some embodiments, antibodies of the invention have:
a) CH3-b of the fusion peptide and CH3-a of the heavy chain have a substitution pair that forms a protrusion-cavity structure, said substitutions comprising, for example, one or more of the substitutions of Table 15, for example one CH3 T366 of the domain is replaced with a relatively large amino acid residue such as tyrosine (Y) or tryptophan (W), and Y407 of the other CH3 domain is replaced with a relatively small amino acid residue such as threonine (T), alanine (A ) or valine (V),
b) CH3-b of said fusion peptide and CH3-a of said heavy chain have a substitution pair that forms an ionic bond, said substitutions comprising, for example, one or more substitutions of Table 16, for example wherein the CH3 domain of comprises one or more substitutions of amino acid residues that are positively charged under physiological conditions, and the other CH3 domain comprises one or more substitutions of amino acid residues that are negatively charged under physiological conditions, said The positively charged amino acid residue is, for example, arginine (R), histidine (H), or lysine (K), and the negatively charged amino acid residue is, for example, aspartic acid (D) or glutamic acid (E). optionally, the substituted amino acid residue is, for example, one or more of D356, L368, K392, D399 and K409;
c) CH3-b of said fusion peptide and CH3-a of said heavy chain have a substitution pair that forms a disulfide bond, said substitutions being, for example, the substitutions of Table 17, and/or d) said fusion CH3-b of the peptide and CH3-a of the heavy chain have substitutions that result in reduced ability to bind protein A, said substitutions being, for example, the substitutions of Table 18, for example one CH3 domain H435 and Y436 of are replaced with arginine and phenylalanine, respectively.

In some embodiments, in the antibodies of the invention, the Fc fragment is CH2 having a sequence of any of SEQ ID NOs: 155-161 or 192, and/or any of SEQ ID NOs: 162-183 CH3 with sequence.

In some embodiments, said heavy chain of the antibody of the invention or the heavy chain of the fusion peptide is a human Fc fragment or a humanized Fc fragment, such as a human IgG Fc fragment, such as IgG1, IgG2, IgG3, IgG4, IgG5 Fc Contains fragments.

In some embodiments, the heavy chain of the antibody of the invention, the heavy chain of the fusion peptide, and/or the Fc fragment of the fusion peptide has a contains one or more substitutions that form a protrusion-void structure pairing between

In some embodiments, the Fc fragment of said heavy chain and/or said fusion peptide of an antibody of the invention comprises one or more substitutions that form a salt bridge pairing between said heavy chain and said fusion peptide. .

In some embodiments, the antibody of the invention comprises Y101, Y102, Y103, Y104, Y105, Y150-8-3, Y150-F8-4, Y150-F8-5, Y150-F8-6, Y150-F8 -7, Y150-F8-8, Y150-F8-9, Y150-F8-10, Y150-F8-11, Y150-F8-12, Y150-F8-13, Y150-F8-14, Y150-F8-15 , Y150-F9-7, Y150-F9-11, Y150-F9-12, MS-hCD3-IC15, MS-hCD3-IC16, MS-hCD3-IC17 and MS-hCD3-IC18;
Here, each component follows the order of fusion peptide VHs-linker1-VLs-hinge1-CH2-CH3-b, heavy chain VHm-CH1-hinge2-CH2-CH3-a, light chain VLm-CL,
Y101 is SEQ ID NO: 45, 129, 63, 142, 159, 167, 106, 154, 139, 159, 166, 107, 148, respectively, or 80%, 85%, 90% with at least one of said amino acid sequences; an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or one with at least one of said amino acid sequences containing amino acid sequences with more than (preferably one or several, more preferably one, two or three) different amino acids,
Y102 is 80%, 85%, 90% with SEQ ID NOs: 48, 129, 63, 142, 159, 167, 106, 154, 139, 159, 166, 107, 148, respectively, or at least one of these amino acid sequences , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or with at least one of these amino acid sequences comprising an amino acid sequence having one or more (preferably one or several, more preferably one, two or three) different amino acids,
Y103 is 80%, 85%, 90% with SEQ ID NOs: 48, 129, 71, 142, 159, 167, 106, 154, 139, 159, 166, 107, 148, respectively, or at least one of these amino acid sequences , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or with at least one of these amino acid sequences comprising an amino acid sequence having one or more (preferably one or several, more preferably one, two or three) different amino acids,
Y104 is 80%, 85%, 90% with SEQ ID NOs: 49, 129, 63, 142, 139, 167, 106, 154, 139, 159, 166, 107, 148, or at least one of these amino acid sequences, respectively , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or with at least one of these amino acid sequences comprising an amino acid sequence having one or more (preferably one or several, more preferably one, two or three) different amino acids,
Y105 is 80%, 85%, 90% with SEQ ID NOs: 49, 129, 71, 142, 139, 167, 106, 154, 139, 159, 166, 107, 148, respectively, or at least one of these amino acid sequences , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or with at least one of these amino acid sequences comprising an amino acid sequence having one or more (preferably one or several, more preferably one, two or three) different amino acids,
Y150-8-3 is SEQ ID NO: 45, 129, 63, 141, 157, 167, 90, 154, 139, 157, 166, 91, 148, respectively, or 80%, 85 with at least one of these amino acid sequences %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-4 is SEQ ID NO: 48, 129, 63, 141, 157, 167, 90, 154, 139, 157, 166, 91, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-5 is SEQ ID NO: 49, 129, 71, 141, 139, 167, 90, 154, 139, 157, 166, 91, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-6 is SEQ ID NO: 51, 129, 71, 141, 139, 167, 90, 154, 139, 157, 166, 91, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-7 is SEQ ID NO: 49, 129, 71, 144, 158, 167, 90, 154, 139, 158, 166, 91, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-8 is SEQ ID NO: 49, 129, 71, 144, 161, 167, 90, 154, 139, 161, 166, 91, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-9 is SEQ ID NO: 49, 129, 71, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-10 is SEQ ID NO: 58, 129, 72, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-11 is SEQ ID NO: 60, 129, 72, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-12 is SEQ ID NO: 60, 129, 73, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-13 is SEQ. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-14 is SEQ ID NO: 61, 129, 73, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or amino acid sequences thereof comprising an amino acid sequence having at least one and one or more (preferably one or several, more preferably one, two or three) different amino acids of
Y150-F8-15 is 62, 129, 73, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148, respectively, or 80%, 85% with at least one of these amino acid sequences; amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or at least these amino acid sequences comprising an amino acid sequence having one and one or more (preferably one or several, more preferably one, two or three) different amino acids,
Y150-F9-7 is 49, 129, 71, 141, 139, 167, 92, 154, 139, 157, 166, 93, 150, respectively, or at least one of these amino acid sequences and 80%, 85%; amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or at least these amino acid sequences comprising an amino acid sequence having one and one or more (preferably one or several, more preferably one, two or three) different amino acids,
Y150-F9-11 is 49, 129, 71, 144, 161, 167, 92, 154, 139, 161, 166, 93, 148, respectively, or at least one of these amino acid sequences and 80%, 85%; amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or at least these amino acid sequences comprising an amino acid sequence having one and one or more (preferably one or several, more preferably one, two or three) different amino acids,
Y150-F9-12 is 49, 129, 71, 144, 192, 167, 92, 154, 139, 192, 166, 93, 148, respectively, or 80%, 85% with at least one of these amino acid sequences; amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or at least these amino acid sequences comprising an amino acid sequence having one and one or more (preferably one or several, more preferably one, two or three) different amino acids,
MS-hCD3-IC15 is 49, 129, 71, 141, 159, 167, 118, 154, 139, 159, 166, 119, 148, respectively, or 80%, 85% with at least one of these amino acid sequences; amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or at least these amino acid sequences comprising an amino acid sequence having one and one or more (preferably one or several, more preferably one, two or three) different amino acids,
MS-hCD3-IC16 is 49, 129, 71, 141, 157, 167, 118, 154, 139, 157, 166, 119, 148, respectively, or 80%, 85% with at least one of these amino acid sequences; amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or at least these amino acid sequences comprising an amino acid sequence having one and one or more (preferably one or several, more preferably one, two or three) different amino acids,
MS-hCD3-IC17 is 49, 129, 71, 141, 161, 167, 118, 154, 139, 161, 166, 119, 148, respectively, or 80%, 85% with at least one of these amino acid sequences; amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or at least these amino acid sequences comprising an amino acid sequence having one and one or more (preferably one or several, more preferably one, two or three) different amino acids,
MS-hCD3-IC18 is 58, 129, 72, 141, 161, 167, 118, 154, 139, 161, 166, 119, 148, respectively, or 80%, 85% with at least one of these amino acid sequences; amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity, or at least these amino acid sequences It includes amino acid sequences having one and more than one (preferably one or several, more preferably one, two or three) different amino acids.
In some embodiments, the invention provides: an antibody or antigen-binding fragment thereof of about ^10-8 M or less, such as about ^10-8 M, ^10-9 M, ^10-10 M or less, or about 100 nM or less, such as about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, Preferably, said antigen-binding fragment is F(ab')2, F(ab) 2, an antibody or antigen-binding fragment thereof selected from Fab', Fab, Fv, Fd, scFv.

In some embodiments, the invention provides polynucleotides encoding the antibodies or antigen-binding fragments thereof described herein.

In some embodiments, the invention provides expression vectors comprising the polynucleotides described herein.

In some embodiments, the invention provides host cells comprising the polynucleotides or expression vectors described herein.

In some embodiments, the invention provides a method of preparing an antibody of the invention, comprising preparing said antibody by introducing a polynucleotide or expression vector described herein into a host cell. .

In some embodiments, the invention provides antibody conjugates comprising an antibody or antigen-binding fragment thereof as described herein and a conjugate moiety conjugated thereto. Preferably, said conjugate moieties are selected from purification tags (eg His tags), cytotoxic agents, detectable labels, radioisotopes, luminescent substances, colored substances, enzymes or polyethylene glycol.

In some embodiments, the invention provides antibody conjugates. Here, the antibody may be conjugated to a therapeutic agent, prodrug entity, peptide, protein, enzyme, virus, lipid, biological response modifier, drug or macrogol.

In some embodiments, the antibody may be linked or fused to a therapeutic agent. Wherein the therapeutic agent includes a radiolabel, an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent (which may be a pharmaceutical or a chemokine), an ultrasound enhancer, Detectable labels such as non-radioactive labels, combinations thereof, and other components of the same type known in the art may also be included.

In some embodiments, the antibody is detectably labeled by conjugating it with a chemiluminescent compound. Then, the presence of the chemiluminescent-labeled antigen-binding polypeptide can be determined by detecting the luminescence generated during the course of the chemical reaction. Particularly useful chemiluminescent labeling compounds include luminol, isoluminose, theromatic acridinium esters, imidazoles, acridinium salts and oxalates.

In some embodiments, the antibody may also be detectably labeled using a fluorescence-emitting metal, such as 152Eu, or other lanthanum-based metal. These metals may be linked to the antibody with metal chelating groups such as diethylene-triaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA). Techniques for linking various groups to antibodies are known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfel. d et al. (ed.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623). -53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications. , Pinchera et al. (eds.), pp. 475-506 (1985); , Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)).

In some embodiments, the invention provides fusion proteins comprising the antibodies or antigen-binding fragments thereof described herein.

In some embodiments, the invention comprises an antibody or antigen-binding fragment thereof, antibody conjugate, or fusion protein described herein, optionally further associated with a pharmaceutically acceptable carrier and/or excipient. Pharmaceutical compositions are provided, including formulations.

In some embodiments, the pharmaceutical composition described herein is in a dosage form suitable for oral administration to the gastrointestinal (GI) tract, preferably said dosage form is a tablet, capsule, pill, is selected from powders, granules, emulsions, microemulsions, solutions, suspensions, syrups and elixirs, or the pharmaceutical composition is subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection , is a dosage form suitable for intralesional injection.

In some embodiments, the invention provides kits comprising the antibodies or antigen-binding fragments thereof, antibody conjugates, or fusion proteins described herein. Preferably, the kit further comprises a second antibody that specifically recognizes an antibody or antigen-binding fragment thereof, antibody conjugate, or fusion protein as described herein. Said second antibody optionally further comprises a detectable label, for example a radioisotope, a luminescent substance, a colored substance, an enzyme.

In some embodiments, the invention provides said antibody or antigen-binding fragment thereof for the treatment of a disease, or an antibody or antigen-binding fragment thereof as described herein for the treatment of a disease. or use of an antibody or antigen-binding fragment thereof as described herein in the preparation of a medicament for the treatment of a disease.

In some embodiments, the antibodies described herein can be used in combination with other therapeutic agents (eg, therapeutic agents for treating tumors or cancer).

In some embodiments, the invention comprises an antibody or antigen-binding fragment thereof described herein, a pharmaceutical carrier, instructions for use, and any other therapeutic agent (e.g., for treating a tumor or cancer). A kit containing the therapeutic agent) is provided.

In some embodiments, in the compositions and/or kits described herein, the antibody or antigen-binding fragment thereof is conjugated with a cytotoxic moiety, enzyme, radioactive compound, cytokine, interferon, target or reporting moiety. gated.

In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat diseases such as cancer or tumors.

In some embodiments, the invention provides the use of said antibodies or antigen-binding fragments thereof to treat diseases such as cancer or tumors.

In some embodiments, the invention provides the use of antibodies or antigen-binding fragments thereof for preparing a medicament for treating diseases such as cancer or tumors.

In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat diseases such as cancer or tumors. Said diseases include, but are not limited to, multiple myeloma, lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma).

In some embodiments, the invention provides methods of humanizing anti-CD3 antibodies, and humanized sequences derived therefrom. Monoclonal antibodies and multifunctional antibodies prepared based on the humanized antibody sequences have suitable affinities, high stability and good cell-killing ability.

In some embodiments, the humanized anti-CD3 antibodies described herein have a higher degree of homology than control antibodies, such as the original antibody SP34, and anti-CD3 antibodies with high homology provided elsewhere. In terms of biological activity and stability of the functional antibody, it exhibits superior biological activity and/or stability to other anti-CD3 antibodies.

In some embodiments, the present invention provides multifunctional antibodies and methods of making. The antibody comprises (a) a light-heavy chain pair and (b) a fusion peptide, wherein the light-heavy chain pair has specificity for a tumor cell or microorganism, and the fusion peptide comprises: An antibody comprising a single chain variable region fragment (ScFv) and an Fc fragment having a CH2 domain and/or a CH3 domain and having specificity for immune cells.

In some embodiments, the light-heavy chain pair or VLm-VHm pairing of an antibody of the invention has specificity for a tumor antigen. In some embodiments, the tumor antigen is selected from PD-L1, SLAMF7, CD38, BCMA, and the like. In some embodiments, the light chain-heavy chain pair or VLm-VHm pairing has specificity for proteins that are overexpressed on tumor cells as compared to corresponding non-tumor cells.

In some embodiments, the light chain-heavy chain pair or VLm-VHm pairing has specificity for viruses or bacteria. In one aspect, said light chain-heavy chain pair or VLm-VHm pairing has specificity for endotoxin.

In some embodiments, the immune cells are T cells, CIK cells, NKT cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, macrophages, natural killer cells, eosinophils, basophils It is selected from spheres and mast cells.

In some embodiments, the ScFv or VLs-VHs pairing comprises an antigen (e.g., CD3, CD4, CD8, CD40L, CD152, CD16, CD56, CD94, CD158, CD161, CD19, CD20, CD21, CD40 ). In some embodiments, the antigen is CD3.

In some embodiments, the light chain is linked to the heavy chain or fusion heavy chain by a disulfide bond. In some embodiments, the heavy chain is attached to the fusion peptide by one or more disulfide bonds. In some embodiments, said fusion heavy chain 1 is linked to said fusion heavy chain 2 by one or more disulfide bonds. In some embodiments, the heavy chain or fusion heavy chain comprises a human Fc fragment or a humanized Fc fragment. In some embodiments, the Fc fragment of the heavy chain or fusion heavy chain comprises a human IgG Fc fragment. In some embodiments, the Fc fragment of said fusion peptide comprises a human Fc fragment or a humanized Fc fragment. In some embodiments, the Fc fragment of said fusion peptide comprises a human IgG Fc fragment.

In some cases, the heavy chain, the fused heavy chain and/or the Fc fragment of the fusion peptide have a protrusion-cavity pairing between the heavy chain and the fusion peptide, compared to a wild-type antibody fragment. contains one or more substitutions that form knobs-into-holes. This pairing can significantly improve the pairing efficiency of the heterodimer of the heavy chain and the fusion peptide.

In some cases, the heavy chain and/or the Fc fragment of the fusion peptide comprises one or more substitutions that form a salt-bridge pairing between the heavy chain and the fusion peptide. This pairing can significantly improve the pairing efficiency of the heterodimer of the heavy chain and the fusion peptide.

In some cases, the CH2 domain of the fusion peptide is located between the scFv fragment and the CH3 domain. In one aspect, the fusion peptide does not contain a CH1 domain.

In one embodiment, the invention further provides a composition comprising the antibody of any of the previous embodiments. In one aspect, the carrier is a pharmaceutical carrier.

In another embodiment, the invention provides a conjugate comprising the antibody of any preceding embodiment that binds to one or more antigens.

FIG. 1A shows a schematic representation of the antibody structure. FIG. 1B shows a schematic representation of the primary protein structure of each component of the antibody. FIG. 2 shows a schematic representation of the primary protein structure of each component of the monoclonal antibody. FIG. 3 shows the expression levels of humanized CD3 monoclonal antibodies. FIG. 4 shows the ability of monoclonal antibodies to bind to CD3 + T cells. FIG. 5 shows expression levels of multifunctional antibodies assembled with humanized anti-CD3 antibodies after transient transfection into CHO cells. FIG. 6 shows the affinity of existing anti-CD3 antibodies, monoclonal antibodies, to cells. Figure 7 shows the ability of various multifunctional antibodies of the invention to activate T cells in vitro. Figure 8 shows the ability of various multifunctional antibodies to kill multiple myeloma cells MC/CAR in vitro. Figure 9 shows the in vitro killing ability of various multifunctional antibodies against lung cancer cell H358. FIG. 10 shows the accelerated thermal stability test of multifunctional antibody Y105 of the invention, comparative antibody Y106, CT-F4, CT-F5, CT-F6 at 40 ^° C. in citrate buffer system. FIG. 11 shows the multifunctional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7, Y150-F8-8 of the present invention, comparative antibodies Y150-F8-1, Y150-F8-2, CT- F1, CT-F2, CT-F3 show accelerated thermal stability studies at 40 ^° C. in citrate buffer system. FIG. 12 shows the multifunctional antibodies Y150-F8-9, F8-10, F8-12, F9-7, F9-11 of the invention, comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2, CT-F3 show accelerated thermal stability test at 40 ^° C in citrate buffer system. FIG. 13 shows that multifunctional antibodies MS-hCD3-IC15, IC16, IC17, IC18 of the invention and comparative antibodies IC-2 through IC-7 underwent an accelerated thermal stability study at 40 ^° C. in a citrate buffer system. show. FIG. 14 shows multifunctional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7, Y150-F8-8 of the present invention, comparative antibodies Y150-F8-1, Y150-F8-2, CT- F1, CT-F2, CT-F3 represent accelerated thermal stability studies at 40 ^° C. in histidine buffer system. FIG. 15 shows multifunctional antibodies Y150-F8-9, F8-10, F8-12, F9-7, F9-11 of the invention, comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2, CT-F3 show accelerated thermal stability test at 40 ^° C in histidine buffer system. Figure 16 shows an accelerated thermal stability study of multifunctional antibodies MS-hCD3-IC15, IC16, IC17, IC18 of the invention and comparative antibodies IC-2 to IC-7 at 40 ^° C in histidine buffer system. . FIG. 17 shows the acid resistance stability test of the multifunctional antibody Y105 of the present invention, comparative antibody Y106, CT-F4, CT-F5 and CT-F6 in a pH 3.5 citrate buffer system. FIG. 18 shows multifunctional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7, Y150-F8-8 of the present invention, comparative antibodies Y150-F8-1, Y150-F8-2, CT- F1, CT-F2, CT-F3 show acid resistance stability test in pH 3.5 citrate buffer system. FIG. 19 shows multifunctional antibodies Y150-F8-9, F8-10, F8-12, F9-7, F9-11 of the invention, comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 show an acid resistance stability test in a pH 3.5 citrate buffer system. Figure 20 shows the acid tolerance test of the multifunctional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 of the invention and the comparative antibodies IC-2 to IC-7 in a pH 3.5 citrate buffer system. . Figure 21 shows the in vivo effects of various multifunctional antibodies and the monitoring of nodule volume in a mouse tumor model. Here, FIG. 21A shows the multifunctional antibody Y150-F8-8 of the invention, FIG. 21B shows the multifunctional antibody F8-9 of the invention, and FIG. 21C shows the multifunctional antibody F9-11 of the invention. The sequences of the anti-CD3 antibodies are both VH2a and VL5, and the sequences of the anti-CD38 antibodies are different from each other.

DEFINITIONS A naturally occurring antibody contains six “complementarity determining regions” or “CDRs” specifically positioned to form an antigen-binding domain. The remaining amino acids of the antigen-binding domain, termed the "framework" regions, have fewer intramolecular variations. The framework region mainly adopts a β-folding sheet structure, while the CDRs form loops. The loop may connect to and become part of the β-folding sheet structure. The framework regions thus form a scaffold that orients the CDRs by intrachain non-covalent interactions. The antigen binding domains formed by the positioned CDRs define surfaces complementary to immunoreactive antigen epitopes. The complementary surface contributes to non-covalent binding of the antibody to its homologous epitope. A person skilled in the art can readily recognize any given heavy or light chain variable region from the amino acids comprising the CDRs and framework regions, respectively ("Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Health and U.S. Department of Health And Human Services, (1983); see Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)).

As used herein, the term "complementarity determining region" ("CDR") refers to the non-contiguous antigen-binding sites present in the variable regions of both heavy and light chain polypeptides. Such specific regions are described by Kabat et al., US Department of Health and Public Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al. MoI. Biol. 196:901-917 (1987). As defined by Kabat and Chothia, CDRs contain amino acid residues, or substructures of amino acids, that overlap when compared to each other. However, all applications of definitions relating to CDRs of an antibody or variant thereof are included within the terms defined and used herein. Given the variable region amino acid sequence of the antibody, those skilled in the art can generally determine which residues comprise a particular CDR.

Kabat et al. also defined a numbering system directed to variable domain sequences that is applicable to any antibody. The "Kabat numbering" system can be used for any variable domain sequence without any doubt by those skilled in the art and without reliance on any experimental data other than the sequence itself. As used herein, "Kabat number" is the numbering system described by Kabat et al. Its contents are described in the US Department of Health and Public Services, "Sequence of Proteins of Immunological Interest" (1983).

The CDR regions described in the Kabat numbering system are as follows. CDR-H1 begins at position approximately amino acid 31 (i.e., approximately nine residues from the first cysteine residue) and ends to the next tryptophan residue, and contains approximately 5-7 amino acids. . CDR-H2 begins at the 15th residue position after the end of CDR-H1 and ends to the next arginine or lysine residue and contains approximately 16-19 amino acids. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2 and includes 3-25 amino acids and ends to a position of the sequence W-G-X-G, in which X is any amino acid. CDR-L1 begins at approximately the 24th residue position (ie, after the cysteine residue) and ends with the next tryptophan residue and contains approximately 10-17 residues. CDR-L2 begins approximately 16 residues after the end of CDR-L1 and contains approximately 7 residues. CDR-L3 begins at approximately the 33rd residue position after the end of CDR-L2 (i.e., after the cysteine residue) and contains about 7-11 residues and has the sequence F or W-G-X. -G, in which X is any amino acid.

The antibodies described herein can be derived from any animal. Said animals include birds and mammals, including primates. Preferably, the antibody is a human, baboon, rhesus, cynomolgus, murine, donkey, rabbit, goat, guinea pig, camel, alpaca, horse or chicken antibody.

The humanized antibodies described herein can specifically bind to CD3, eg, primate CD3, wherein said primate CD3 includes, eg, human and/or monkey CD3.

As used herein, the term "heavy chain constant region" includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region has at least any of a CH1 domain, a hinge (e.g., upper hinge region, middle hinge region, and/or lower hinge region) domain, CH2 domain, CH3 domain, or variants or fragments thereof. including. For example, antigen-binding polypeptides used in the present invention include a polypeptide chain having a CH1 domain; a polypeptide having a CH1 domain, at least a portion of a hinge domain and a CH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; A polypeptide chain having a domain, at least a portion of a hinge domain and a CH3 domain, or a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, a CH2 domain, a CH3 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain with a CH3 domain. Antibodies of the invention may also lack at least a portion of the CH2 domain (eg, all or part of the CH2 domain). It should be understood by those of ordinary skill in the art that heavy chain constant regions may be modified to differ in amino acid sequence from naturally occurring immunoglobulin molecules.

The heavy chain constant regions of the antibodies described herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a CH1 domain from an IgGl molecule and a hinge region from an IgG3 molecule. In another form, the heavy chain constant region may comprise a hinge region that is partly from an IgGl molecule and partly from an IgG3 molecule. In another form, the heavy chain portion may comprise a chimeric hinge partly from an IgGl molecule and partly from an IgG4 molecule.

The term "light chain constant region" as used herein comprises amino acid sequences derived from antibody light chains. Preferably, said light chain constant region comprises at least one of a kappa constant domain and a lambda constant domain.

A "light-heavy chain pair" refers to a collection of light and heavy chains. Light and heavy chains can form dimers through disulfide bonds between the CL and CH1 domains of the light chains.

The subtile and three-dimensional structures of constant regions of various immunoglobulin classes are known. The term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain. The term "CH1 domain" comprises the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is proximal to the VH domain and is amino terminal to the hinge region of immunoglobulin heavy chain molecules.

As used herein, the term "CH2 domain" includes a portion of the heavy chain molecule. Such moieties range, for example, from approximately residue 244 to residue 360 of the antibody in the common numbering scheme (residues 244 to 360 in the Kabat numbering system, EU corresponding to residues 231-340 in the numbering system, see Kabat et al., US Department of Health and Public Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique in that it is not tightly paired with other domains. Instead, both N-linked side chain carbohydrate chains are inserted between both CH2 domains of an intact native IgG molecule. The literature describes that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible enough to allow independent movement of both N-terminal antigen binding regions. The hinge region can be divided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol, 161:4083 (1998)).

As used herein, the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine contains a sulfhydryl group that can form a disulfide bond or crosslink with another sulfhydryl group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and both heavy chains are located at positions 239 and 242, Kabat numbering system (226 or 229, EU numbering system). position) are linked by both disulfide bonds.

As used herein, the term "chimeric antibody" means an antibody in which the immunoreactive region or site is derived from or derived from a first species; In addition, its constant region (the constant region may be complete, partial, or modified according to the present invention) is derived from the second species. In some embodiments, the target binding region or site is non-human (eg, mouse or primate) and the constant region is human.

As used herein, "humanization percentile" measures the number of in-frame amino acid difference values (i.e., non-CDR difference values) between a humanized domain and a domain of that species, and Calculated by subtracting from the total number of amino acids divided by the total number of amino acids and multiplying by 100.

The term "specifically binds" or "has specificity for" generally refers to the binding of an antibody through its antigen-binding domain to an antigen epitope such that the binding binds the antigen-binding domain to the antigen epitope. It means that there is always a certain complementarity between By this definition, an antibody that, when bound to that antigenic epitope, is more readily bound by its antigen-binding domain than it is bound to a random, irrelevant antigenic epitope, is said to "bind specifically" to an antigenic epitope. . As used herein, the term "specificity" is used to determine the affinity with which an antibody binds to a particular antigenic epitope. For example, antibody "A" may be considered to have higher specificity for a particular antigenic epitope than antibody "B". Alternatively, an antibody "A" may be said to have greater specificity in binding to the correlated antigenic epitope "D" compared to the antigenic epitope "C". In some embodiments, the antibodies of the invention bind to a target with a KD of less than about ^10-8 M, such as less than about ^10-8 M, ^10-9 M, ^10-10 M or less. In some embodiments, antibodies of the invention are less than about 100 nM, such as about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM , 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less and binds to the target with an EC50 of less than or equal to.

It should be understood that an entity refers to one or more (species) of such entities, where there is no express limitation in number. For example, "multifunctional antibody" is understood to refer to one or more (species) of multifunctional antibodies. Similarly, the terms "one or more" and "at least one" can be used interchangeably herein, where there is no explicit numerical limitation.

As used herein, the term "polypeptide" includes not only the singular "polypeptide" but also the plural "polypeptide" and also includes linear bonds through amide bonds (also called peptide bonds). A molecule consisting of monomers (amino acids) that are linked together. The term "polypeptide" refers to a chain or chains of two or more amino acids, and does not imply a specific length. Thus, peptides, dipeptides, terpeptides, oligopeptides, "proteins", "chains of amino acids", or other terms referring to any chain or chains consisting of two or more amino acids are all referred to as "polypeptides". included in the definition of "peptide". Also, the term "polypeptide" can be used interchangeably or interchangeably with these terms. The term "polypeptide" refers to the products of post-expression modifications of polypeptides, including glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, hydrolytic dissolution of proteins, Or, including but not limited to modifications of non-naturally occurring amino acids. A polypeptide may be derived from a naturally occurring organism or produced by homologous recombination techniques, but is not necessarily translated from a designated nucleic acid sequence. They can be made by any method, including chemical synthesis.

As used herein, the term "isolated" with respect to cells, nucleic acids (e.g., DNA or RNA) refers to molecules that are isolated from other naturally occurring large molecules, DNA or RNA, respectively. . As used herein, the term "isolated" also includes nucleic acids or peptides substantially free of cellular, viral or media material when produced by recombinant DNA techniques, or produced by chemical synthesis Occasionally refers to substantially free of chemical precursors or other chemicals. "Isolated nucleic acid" also refers to nucleic acid fragments not found in nature, including non-naturally occurring fragments. As used herein, a cell or polypeptide that is otherwise isolated from a cellular protein or tissue from the term "isolated". An isolated polypeptide includes purified or recombinant polypeptides.

As used herein, the term "recombinant," when referring to a polypeptide or polynucleotide, refers to forms of the polypeptide or polynucleotide that do not occur in the natural state. Here, one non-limiting example can be achieved by combining polynucleotides or polypeptides that do not normally exist together.

"Homology" or "identity" or "similarity" refers to the degree of sequence similarity between two peptide chain molecules or between two nucleic acid molecules. Homology can be measured by comparing each sequence position. Comparisons can be made by alignment. If the same base or amino acid is present at a position in the compared sequences, the molecules at that position are considered homologous. The degree of homology between sequences is a function of the number of pairings, or sites of homology, shared by the sequences. A "non-related" or "non-homologous" sequence refers to having less than 40% homology, preferably less than 25% homology, with one of the sequences of the invention.

Polynucleotides or polynucleotide regions (or polypeptides or polypeptide regions) are conjugated to one other sequence at a given percentile (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%) %, 95%, 98% or 99%) “sequence identity” means that when the two sequences are compared when aligned, they have similar bases (or amino acids) at that percentile. are doing. Alignments, percentile homology or sequence identity as described above can be determined by software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). Preferably, the alignment uses default parameters. Biologically equivalent polynucleotides refer to polynucleotides that have the specified percentiles of homology as described above while simultaneously encoding the same or similar biologically active polypeptides.

The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid whose nucleotide sequence has a given degree of homology or sequence identity with the nucleotide sequence of said nucleic acid or its complementary sequence. A homologue of a double-stranded nucleic acid refers to a nucleic acid containing a specific nucleotide sequence having a given degree of homology with another nucleic acid or its complementary sequence. In one aspect, a homologue/homologue of a nucleic acid is one that is capable of hybridizing to that nucleic acid or its complementary sequence. Similarly, "equivalent polypeptide" refers to a polypeptide having a given degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some cases, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some cases, the equivalent sequence retains the activity (eg, ability to bind to an antigenic epitope) or structure (eg, salt bridge) of the reference sequence.

Hybridization reactions may be performed under different "stringent" conditions. Generally, less stringent hybridization reactions are performed at about 40° C. in solutions of about 10×SSC, or equivalent ionic strength/temperature. Moderate stringency hybridizations are typically performed at about 50° C. and about 6×SSC, and high stringency hybridization reactions are typically performed at about 60° C. and about 1×SSC. will be Hybridization reactions may be performed under "physiological conditions", which are well known to those skilled in the art. One non-limiting example of physiological conditions is normal temperature, ionic strength, pH value, Mg2+ concentration in the cell.

Polynucleotides are composed of specific sequences of four nucleotide bases: adenine (A), cytosine (C), guanine (G) and thymine (T). When the polynucleotide is RNA, thymine is replaced with uracil (U). As such, the term "polynucleotide sequence" is an ordered letter representation of an arrangement of polynucleotide molecules. The ordered letter representation can be entered into a database for a computer with a central processing unit for use in applications in bioinformatics, eg functional genomics, homology searching. The term "polymorphism" refers to the coexistence of one or more genes or portions thereof. When a portion of a gene has at least two different types, ie two different nucleotide sequences, that portion of the gene is called a "polymorphic region of the gene." A polymorphic region may be a mononucleotide with different identities in different alleles.

The terms "polynucleotide" and "oligonucleotide" can be used interchangeably and refer to polymeric forms of nucleotides of any length. The polymeric form of the nucleotides may be deoxyribose nucleotides, ribose nucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may have any known or unknown function. Non-limiting examples of polynucleotides include genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, Examples include siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA having a single arbitrary sequence, RNA having a single arbitrary sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides or nucleotide analogs. When the modified nucleotides are present, the base structure is modified before or after assembly of the polynucleotide. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, eg, conjugated by a labeling moiety. The term also refers to double- and single-stranded molecules. Each embodiment of the polynucleotides of the present invention includes the double-stranded form, the two known complementary single-stranded forms, and the double-stranded form, unless otherwise indicated or claimed. Including what you would expect.

As applied to a polynucleotide, the term "encoding" refers to a polynucleotide that is believed to "encode" a particular polypeptide. The polynucleotide can be transcriptome and/or translated to produce mRNA and/or fragments thereof of the polypeptide in its native state or if manipulated by methods known to those of skill in the art. The antisense strand is the complement of such nucleic acid. The coding sequence can be derived from the antisense strand.

As used herein, the term "detectable tag" refers to a directly or indirectly detectable compound or composition. The compound or composition can be conjugated, directly or indirectly, to a composition (e.g., polynucleotide or protein, including antibodies) to be detected, resulting in a "tagged" composition. . The term also refers to a sequence that binds to the polynucleotide that, upon expression of the inserted sequence, provides a signal, such as green fluorescent protein (GFP). Such tags are themselves detectable (eg, radioisotope tags, fluorescent tags) or enzymatic tags capable of catalyzing a detectable chemical alteration of a substrate compound or composition. The tags can be used for small scale assays, but are more suitable for high flux screening. Similarly, suitable tags include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, proteins (including enzymes). Such tags may be only detectable or may additionally be quantifiable. Detectable-only reactions typically include reactions whose presence can only be confirmed. As used herein, quantifiable reactions typically include reactions that have quantifiable (eg, numerically reportable) values of intensity, polarization, and/or other properties. In luminescence or fluorescence analysis, the detectable reaction may directly use a luminescent or fluorescent group that is actually associated with the analyte, but indirectly another component (e.g. A luminescent or fluorescent group linked to a reporter molecule or indicator) may also be used.

As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a whole antibody or any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing molecule that contains at least a portion of an immunoglobulin molecule that has antigen-binding biological activity. Examples of such embodiments include complementarity determining regions (CDRs) of heavy or light chains or ligand binding portions thereof, variable regions of heavy or light chains, constant regions of heavy or light chains, frame (FR) ) region or any portion thereof, or at least a portion of the binding protein.

The term "antibody fragment" or "antigen-binding fragment" as used herein is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and so on. Antibody fragments that bind the same antigen, regardless of structure, are considered intact antibodies. The term "antibody fragment" includes aptamers, enantiomers and dimers of aptamers. The term "antibody fragment" also includes synthetic or genetically engineered proteins that, like antibodies, are capable of binding a specific antigen to form a complex.

"Single-chain variable region fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some cases, these regions are joined by short linker peptides having 10 to about 25 amino acids. The linker may be rich in glycine for flexibility, or may contain serine or threonine for solubility. Also, the N-terminus of VH can be linked to the C-terminus of VL, or vice versa. The protein has only the constant region removed and a linker introduced, retaining the properties of the original immunoglobulin. ScFv molecules are known in the art and include those described in US Pat. No. 5,892,019.

Heavy chains, as should be understood by those skilled in the art, are divided into gamma, mu, alpha, delta, and epsilon (γ, μ, α, δ, ε), with several subclasses (eg, γl-4). have. The "class" of an antibody, eg, IgG, IgM, IgA, IgG, or IgE, is determined by the properties of the chain. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and functionally specific. All modified forms of these classes and isotypes can be readily discerned by those skilled in the art, upon reference to the specification, and thus are within the scope of the present invention. All immunoglobulin classes are clearly within the scope of this application, and the following description is generally directed to the IgG class of immunoglobulin molecules. IgG is a standard immunoglobulin molecule consisting of two identical light chain polypeptides (both having a molecular weight of about 23,000 Daltons) and two identical heavy chain polypeptides (both having a molecular weight of 53,000 Daltons). ,000-70,000). These four chains are typically linked in a "Y" configuration by disulfide bonds. Here the light chain supports the heavy chain from the mouth of the "Y" structure and is further extended through the variable region.

Antibodies, antigen-binding polypeptides, variants or derivatives thereof of the invention may be polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric, single chain Antibodies, antigen-epitope binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), VL domains or VH domains, fragments generated by Fab expression libraries, anti-idiotypic (anti-Id) antibodies (e.g., including anti-Id to LIGHT antibodies disclosed herein), but It is not limited to these. The immunoglobulin or antibody molecules of the invention may be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), any class of immunoglobulin molecule (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclasses.

Light chains are divided into kappa or lambda (κ, λ). Each class of heavy chain can bind either a kappa or a lambda light chain. At all times, a light chain is covalently linked to a heavy chain. Furthermore, when these immunoglobulins are produced by hybridomas, B-cells or genetically modified host cells, the "tail" portions of the two heavy chains are held together by covalent, disulfide or non-covalent bonds. ing. In the heavy chain, the amino acid sequence extends from the N-terminus of the forks of the Y-shaped structure to the bottom C-terminus of each chain.

Both light and heavy chains are divided into structural and functional homologous regions. The terms "constant" and "variable" are used for functional purposes. Here, it should be recognized that the light chain variable domain (VL) together with the heavy chain variable domain (VH) determine antigen recognition and specificity. On the other hand, the light chain constant domain (CL) and the heavy chain constant domain (CH1, CH2 or CH3) are important for e.g. secretion, transplacental mobility, Fc receptor binding, complement binding, etc. Provides biological properties. In general, the number of constant region domains increases with increasing distance from the antigen-binding site or amino-terminal position of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL domains actually comprise the carboxy-termini of the heavy and light chains, respectively.

As noted above, the variable region enables the antibody to selectively recognize and specifically bind antigenic epitopes of antigens. That is, the VL and VH domains of an antibody, or subclasses of complementarity determining regions (CDRs), combine to make up the variable regions that define the three-dimensional antigen-binding site. The tetravalent antibody structure forms the antigen binding site. The antigen binding site is present at the end of each arm of the Y shape. More specifically, the antigen-binding site is defined by three CDRs of each VH and VL chain (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). It is In some instances, for example, immunoglobulins derived from or modified based on an immunoglobulin, the complete immunoglobulin molecule may be composed only of heavy chains and no light chains. . See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

As used herein, the term "treatment" refers to a therapeutic treatment and prophylaxis, or prophylactic-curative treatment that prevents or slows (reduces) the progression of an undesirable physiological change or disease in a subject, such as cancer. means Beneficial or desired clinical outcomes, whether detectable or not, include relief of symptoms, reduction in disease stage, stabilization of disease state (e.g., preventing it from exacerbating), slowing or delaying disease progression, It includes, but is not limited to, amelioration or alleviation of a disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Conditions requiring treatment include those that already have the disease or condition, those that are predisposed to the disease or condition, or those that prevent the disease or condition.

As used herein, "subject", "individual", "animal", "patient" or "mammal" means any subject, particularly a mammalian subject, in need of diagnosis, prognosis or treatment. Mammalian subjects include humans, domesticated animals, farm animals, zoos, playgrounds, or companion animals such as dogs, cats, guinea pigs, rabbits, rats, mice, rodents, horses, cows, cows, primates (e.g. , humans, monkeys such as cynomolgus monkeys and rhesus monkeys, baboons, chimpanzees, etc.).

As used herein, the terms e.g., "patient in need of treatment" or "subject in need of treatment" refer to patients who benefit from administration, e.g., testing, diagnostic procedures and/or treatment of the antibodies and compositions of the invention. including mammalian subjects, such as humans, who receive

Multifunctional Antibodies One embodiment of the invention provides heterodimeric antibodies composed of two different antigen-binding polypeptide units. In some cases, the heterodimer differs in size from its corresponding homodimer. Utilizing this size difference can contribute to the separation of heterodimers and homodimers.

In some cases, as in Figure 1, one of the two antigen-binding polypeptide units comprises a light-heavy chain pair that resembles a wild-type antibody. Throughout the specification such units are also referred to as "monovalent units".

In some cases, as in Figure 1, the other antigen binding polypeptide unit comprises a single chain variable region fragment (scFv). Such scFv can be fused to the N-terminus of the constant fragment (Fc) of an antibody, termed a fusion peptide. Throughout the specification this fusion peptide is also referred to as a "single chain unit".

The present invention provides multifunctional antibodies and methods of manufacture, wherein the antibodies comprise (a) a light-heavy chain pair with specificity for tumor cells, (b) a single chain variable region fragment (ScFv) and an immune cell-specific fusion peptide comprising an Fc fragment with a CH2 domain and/or a CH3 domain. Said antibodies are called multifunctional antibodies.

Any of the foregoing antibodies or polypeptides may also contain additional polypeptides. Additional polypeptides include, for example, a polypeptide as described herein, a signal peptide of the constant region of an antibody to direct secretion, or other heterologous polypeptides as described herein.

As one of ordinary skill in the art should understand, antibodies derived from the naturally occurring binding polypeptides described herein may differ in amino acid sequence from these naturally occurring binding polypeptides. may be modified. For example, a polypeptide or amino acid sequence from a particular protein may be similar to the starting sequence, eg, have a certain percentage of identity with the starting sequence. For example, it can have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the starting sequence.

Nucleotide or amino acid substitutions, deletions, or insertions may also be made to make conservative substitutions or changes to "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence derived from a particular protein may have one or more independent amino acid substitutions, insertions or deletions, e.g., 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 15, 20 or more independent amino acid substitutions, insertions or deletions, but can be identical to the starting sequence. In some embodiments, a polypeptide or amino acid sequence derived from a particular protein has 1-5, 1-10, 1-15, or 1-20 independent amino acid substitutions, insertions, or deletions from the starting sequence. have a loss.

In some embodiments, an antigen-binding polypeptide comprises an amino acid sequence or one or more groups that do not generally bind to an antibody. Exemplary modifications are described in more detail below. For example, single-chain Fv antibody fragments of the invention may comprise flexible linker sequences or may be modified to add functional groups (e.g., polyethylene glycol (PEG), drugs, toxins or labels). good too.

Antibodies, variants or derivatives thereof of the present invention include modified derivatives. That is, any type of molecule that is covalently linked to the antibody and that covalent bond does not interfere with the antibody's binding to the antigenic epitope. For example, without limitation, the antibody may be glycosylated, acetylated, pegylated, phosphorylated, phosphorylated, amidated, derivatized by known protecting/cleavage groups, dissolved by proteolysis, etc. , cellular ligands or linkage to other proteins. Any of a number of chemical modifications can be made by known techniques. Such techniques include, but are not limited to, specific chemical lysis, acetylation, formylation, tunicamycin metabolism synthesis, and the like. Additionally, the antibody may comprise one or more non-classical amino acids.

In other embodiments, antigen-binding polypeptides of the invention may contain conservative amino acid substitutions.

A "conservative amino acid substitution" is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, threonine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Therefore, nonessential amino acid residues in immunoglobulin polypeptides are preferably replaced with other amino acid residues from the same side chain family. In another embodiment, contiguous amino acids can be replaced with structurally similar contiguous amino acids that differ in order and/or side chain family composition.

Methods of Producing Antibodies Methods of producing antibodies are known techniques in the art and are described herein. In some embodiments, the variable and constant regions of the antigen-binding polypeptides of the invention are fully human. Fully human antibodies can be prepared by techniques described in the prior art and as described in this application. For example, a fully human antibody against a particular antigen is prepared by administering the antigen to a genetically modified animal that has been modified to produce an antibody that responds to this type of antigenic stimulation and whose endogenous locus has been disallowed. be able to. Exemplary techniques by which such antibodies can be produced are described in US Pat.

In some embodiments, the antibodies prepared do not elicit an adverse immune response in the treated animal (eg, human). In one embodiment, the antigen-binding polypeptide, variant or derivative thereof of the invention has been modified by techniques known in the art to reduce its immunogenicity. For example, antibodies can be humanized, primatized, deimmunized, or chimerized. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies. Said antibodies retain or to a greater extent retain the antigen-binding properties of the parent antibody, but are less immunogenic in the human body. This can be accomplished in several ways: (a) grafting of entire non-human variable domains onto human constant regions to create chimeric antibodies; (b) one or more non-human complementarity determination grafting at least part of the regions (CDRs) onto human framework and constant regions, optionally retaining important frame residues, or (c) grafting entire non-human variable domains; , by substitution of surface residues to "cover" it with human-like moieties.

Deimmunization can be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes altering T cell antigen epitopes by altering antibodies (see, e.g., International Application Publication Nos. WO/9852976 A1 and WO/0034317 A2 ). For example, the present application analyzes the variable heavy and variable light chain sequences from the starting antibody and creates a "map" of human T cell antigen epitopes from each V region. The map shows the location of antigenic epitopes in relation to complementarity determining regions (CDRs) and other important residues in the sequence. From this T cell antigen epitope map, analysis of single T cell epitopes can identify alternative amino acid substitutions with low risk of altering the activity of the final antibody. The present application has designed a series of selectable variable heavy and light chain sequences, including combinations of amino acid substitutions, which are then incorporated into a series of binding polypeptides. Typically, 12-24 antibodies are generated and tested for binding and/or functionality. Whole antibodies are then produced by cloning complete heavy and light chain genes, including modified variable regions and human constant regions, into expression vectors and introducing the resulting plasmids into cell lines. These antibodies are then compared by appropriate biopsy and bioassays to identify the best variables.

Binding specificity of antigen-binding polypeptides of the invention can be determined in in vitro experiments such as, for example, immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

For the preparation of single-chain units of the invention, (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883). (1988); Ward et al., Nature 334:544-554 (1989)) can be used. A single chain fusion peptide is obtained by joining the heavy and light chain fragments of the Fv region via an amino acid bridge to form a single chain unit. Techniques for synthesizing functional Fv fragments in E. coli (Skerra et al., Science 242:1038-1041 (1988)) can also be used.

Examples of techniques used to produce single-chain Fvs (scFvs) and antibodies are U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For certain applications involving the use of antibodies in humans, in vitro assays, chimeric, humanized or human antibodies are preferred. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, eg, an antibody comprising a variable region from a murine monoclonal antibody and the constant regions of a human immunoglobulin. Methods for making chimeric antibodies are known in the art. See, eg, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715;

A humanized antibody is an antibody molecule that binds to a desired antigen from a non-human species, the antibody molecule comprising one or more complementarity determining regions (CDRs) from the non-human species and a human immunoglobulin molecule. It has the framework region from which it was derived. In general, frame residues in the human framework regions are preferably modified to increase antigen-binding ability by substituting the corresponding residues from the CDR donor antibody. These frame substitutions can be recognized by methods known in the art. For example, establishing an interaction model of CDRs and frame residues identifies frame residues that are relatively important for antigen binding and sequence, and determines unusual frame residues at specific positions (see, for example, Queen et al. , U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which is incorporated by reference in its entirety into this application). Antibody humanization uses several techniques known in the art. Such techniques include, for example, CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; US Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering, ) or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994)). USA 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332, incorporated by reference in its entirety into this application); can be mentioned.

Completely human antibodies are particularly preferred for treatment of human patients. Human antibodies can be prepared by a number of methods known in the art including bacteriophage display methods using antibody libraries derived from human immunoglobulin sequences. Also, U.S. Patent Nos. 4,444,887 and 4,716,111; /33735 and WO 91/10741, each of which is incorporated by reference into this application.

Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins and capable of expressing human immunoglobulin genes. For example, complexes of human heavy and light chain immunoglobulin genes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions and multivariable regions may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. Alternatively, human immunoglobulin loci may be non-functionally added to the mouse heavy and light chain immunoglobulin genes by homologous recombination, respectively, or introduced simultaneously. Specifically, homozygous deletion of the JH region inhibits endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized with the selected antigen in the usual manner, eg, with all or part of the desired target polypeptide. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice by standard hybridoma technology. The edited human immunoglobulin genes in the transgenic mice are rearranged during B-cell differentiation, followed by class switching and somatic mutation. Therefore, the technology can be used to produce IgG, IgA, IgM and IgE antibodies that are therapeutically useful. An overview of such techniques for producing human antibodies is provided by Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). Detailed descriptions of the techniques used to produce human antibodies and human monoclonal antibodies, and the technical configurations used to produce these antibodies, can be found, for example, in PCT Publication Nos. WO 98/24893; WO 96/34096; WO 96/33735; 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; , which are incorporated by reference into this application. Also, companies such as Abgenix, Inc. (Freemont, Calif.) and GenPharm ((San Jose, Calif.), for example, can provide human antibodies to selected antigens obtained with techniques similar to those described above.

Fully human antibodies which recognize selected antigenic epitopes may be produced using a technique called "guided selection". In the method, a non-human monoclonal antibody of choice, such as a murine antibody, guides the selection of fully human antibodies that recognize the same antigenic epitope (Jespers et al., Bio/Technology 72:899-903 (1988). See US Pat. No. 5,565,332, which is hereby incorporated by reference in its entirety).

In some embodiments, the DNA encoding the desired monoclonal antibody is obtained by routine methods (eg, using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of a murine antibody). Easily isolated and sequenced. A preferred source of the DNA is single or subcloned hybridoma cells. The isolated DNA may be inserted into an expression vector. The vectors are then transformed into prokaryotic or eukaryotic host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 cells or myeloma cells. These cells do not produce immunoglobulins. More specifically, the constant region can be synthesized using a single DNA (which can be synthesized as described herein) as in Newman et al., US Pat. No. 5,658,570, filed Jan. 25, 1995. and variable region sequences can be cloned to prepare antibodies, which are incorporated herein by reference. Essentially, this entails extracting RNA from selected cells, transforming it into cDNA, and amplifying it by PCR using Ig-specific primers. Suitable primers that can be used for this purpose are also described in US Pat. No. 5,658,570. As discussed in more detail below, transformed cells expressing the desired antibody can be cultured in relatively large quantities to provide immunoglobulins for clinical and commercial applications.

Alternatively, one or more CDRs of the antigen-binding polypeptide of the invention can be inserted into the framework regions by conventional recombinant DNA techniques. For example, a non-human antibody is humanized by inserting human framework regions. The framework regions may be naturally occurring or shared framework regions, preferably human framework regions (see, for example, Chothia et al., J. Mol. Biol. 278:457-479 ( 1998), a list of human framework regions). Preferably, the polynucleotide resulting from the combination of the framework regions and CDRs encodes a polypeptide that specifically binds to at least one antigenic epitope (eg, LIGHT) of the desired polypeptide. Preferably, one or more amino acid substitutions may be made within the framework regions. Here, preferably, the amino acid substitution improves the antigen-binding ability of the antibody. The method also allows for amino acid substitutions or deletions to one or more variable region cysteine residues, which are cysteine residues involved in intrachain disulfide bond formation. Antibody molecules that lack one or more intrachain disulfide bonds are then obtained. Any other changes made to the polynucleotide are within the scope of the present application as well as within the prior art.

There is also a technique for producing a "chimeric antibody" by gene splicing a combination of a mouse-derived antibody molecule having appropriate antigen specificity and a human antibody molecule gene having appropriate bioactivity (Morrison et al., Proc. USA: 851-855 (1984); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) can also be used. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, eg, an antibody comprising a murine monoclonal antibody variable region and a human immunoglobulin constant region.

However, another efficient method used to produce recombinant antibodies is disclosed by Newman, Biotechnology 10:1455-1460 (1992). Specifically, the technology results in primatized antibodies that contain monkey variable domains and human constant sequences. That document is incorporated into this application by reference. This technology is also described in co-assigned US Pat. Nos. 5,658,570, 5,693,780 and 5,756,096, each of which is incorporated herein by reference.

In some embodiments, antibody-producing cell lines may be selected and cultured by techniques well known to those of skill in the art. Such techniques are described in various laboratory manuals and primary publications. In this regard, techniques suitable for use herein are described in Current Protocols in Immunology, Coligan et al., eds., Green Publishing Associates And Wiley-Interscience, John Wiley and Sons, New York (1991), as follows: , which is incorporated by reference into this application, including supplementary references.

In some embodiments, criteria techniques known to those of skill in the art can be used to mutate the nucleotide sequences encoding the antibodies of the invention. Such mutations include, but are not limited to site-directed mutagenesis and PCR-mediated mutations that produce amino acid substitutions. Preferably, in said variants (including derivatives), CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2 or CDR-L3 are relative to the variable heavy chain region of reference less than 50 amino acid substitutions less than 40 amino acid substitutions less than 30 amino acid substitutions less than 25 amino acid substitutions less than 20 amino acid substitutions less than 15 amino acid substitutions less than 10 amino acid substitutions 5 Encode less than 1 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions. , or may be randomly mutated along all or part of the coding sequence. For example, when using saturation mutagenesis, the resulting mutants can be screened for bioactivity to ensure that they possess activating mutations.

Therapeutic and Diagnostic Methods The antigen-binding polypeptides, variants or derivatives of the invention can be used in certain therapeutic and diagnostic methods related to cancer or infectious diseases.

This application also relates to treatment with antibodies. The treatment includes administering a bispecific of the present application to a patient, eg, animal, mammal, human, to treat one or more of the diseases or conditions described herein. Therapeutic compounds of the present application may be antibodies of the present application (including variants, derivatives thereof described in the present application) and nucleic acids, or polynucleotides encoding the antibodies of the present application (mutants thereof described in the present application). (including derivatives), but are not limited to these.

Antibodies of the present application can be used to treat, suppress or prevent diseases, conditions or conditions, including malignant diseases or conditions, or conditions associated with such diseases or conditions, e.g., diseases associated with immune responses. In some embodiments, the antibodies of the invention can be used as immunosuppressants. In some embodiments, the antibodies of the invention can be used to treat autoimmune diseases. The antigen-binding polypeptides, variants or derivatives thereof of the present application are used to inhibit the growth, progression and/or growth of cancers, particularly those mentioned above or in the following paragraphs.

Other diseases or conditions associated with increased cell viability can be treated, prevented, diagnosed and/or predicted by the antibodies or variants or derivatives thereof of the present application. Said other diseases or conditions include cancer or tumor progression and/or metastasis, including malignancies, and related diseases such as multiple myeloma, lung cancer (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous). epithelial cancer) and the like, but are not limited to these.

The specific dosage and therapeutic regimen to be used for any particular patient will depend on the specific antigen-binding polypeptide, variant or derivative thereof being used, the patient's age, weight, general health status, sex. It depends on a variety of factors, including diet, intake and administration time, excretion rate, drug combination, and severity of the specific disease being treated. Judgment of these factors by medical caregivers is within the ordinary skill in the art. The amount used will also depend on the individual patient to be treated, the route of administration, the type of formulation, the properties of the compound used, the severity of the disease and the effect desired. Dosages can be determined by pharmacological and pharmacokinetic principles known in the art.

Methods of administering antigen-binding polypeptides, variants or derivatives thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The antigen-binding polypeptides or compositions may be administered by any convenient route, such as infusion or bolus injection, or absorption by epithelial, mucosal or skin linings (eg, oral mucosa, rectal and intestinal mucosa, etc.). can do. It may also be co-administered with other bioactive agents. Therefore, pharmaceutical compositions comprising the antigen-binding polypeptides of the present application can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., powders, ointments). , drops or transdermal patches), sublingual/buccal administration, or as an oral or nasal spray.

The term "parenteral" as used herein refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Administration may be systemic or local. The antibodies of the present application may also need to be introduced into the central nervous system by any suitable route, including intrathecal and intrathecal injection. A ventricular catheter, for example, can contribute to ventricular infusion by connecting the catheter to a reservoir. Pulmonary administration may also be provided, for example, by an inhaler or atomizer, or a formulation containing a nebulizer formulation.

It may also be necessary to administer the antigen-binding polypeptides or compositions of the present application locally to the area in need of treatment. This can be achieved, for example, by local perfusion or topical application during the course of surgery, such as, but not limited to, combination with post-operative wound dressings, injections, catheters, suppositories, or implants. . The implants may be porous, non-porous, or gel-like materials, including films or fibers. Preferably, when administering proteins (including antibodies) of the present application, care should be taken to use materials that do not absorb the proteins.

The dose of the antibody of the present application is an effective amount for treating, suppressing and preventing inflammation, immune or malignant disease, disease or condition that can be determined according to standard clinical techniques. Also, the amount used can be arbitrarily selected to facilitate determination of the optimal dose range in In Vitro experiments. The precise dosage to be prescribed will depend on the route of administration and the severity of the disease, condition or condition, and should be determined according to the judgment of the physician and the specific circumstances of each patient. Effective doses may be extrapolated from dose-response curves derived from In Vitro or animal model test systems.

One embodiment of the invention relates to a method of treating an infectious or malignant disease, condition or condition comprising administering an antibody of the present application, a variant of said antibody. The treatments are usually tested in vitro and then in vivo in acceptable animal models for the desired therapeutic or prophylactic activity before use in humans. Suitable animal models, including genetically modified animals, are known to those of ordinary skill in the art. For example, in vitro assays for demonstrating therapeutic efficacy of the antigen-binding polypeptides described herein include the effect of antigen-binding polypeptides on cell lines or patient tissue specimens. The effects of antigen-binding polypeptides on cell lines and/or tissue specimens can be measured by techniques known to those skilled in the art. These techniques are, for example, analyzes presented by other parts of this application. According to the present application, specimens of patient tissue are cultured in culture and exposed to or otherwise administered compounds to determine whether administration of a particular antigen-binding polypeptide is indicated. In vitro assays, including in vitro cell culture assays, can be used to examine the effect of the compound on said tissue specimen.

Structural Information of Antibodies The structure of a monoclonal antibody is a symmetrical monospecific antibody that has both identical light chains and both identical heavy chains and contains both light-heavy chain pairings and heavy-heavy chain pairings. , where the light-heavy chain pairing targets the same target.

The structure of the multifunctional antibody is an asymmetric bispecific antibody having a light chain, a heavy chain and a fusion peptide, comprising a light-heavy chain pairing and a heavy chain-fusion peptide pairing, wherein Chain pairing targets a tumor antigen and the fusion peptide ScFv targets the immune cell antigen CD3.

In some cases, the heavy chain is linked to the fusion peptide by one or more disulfide bonds, or one or more disulfide bonds are formed between two different fusion heavy chains. . In one aspect, the one or more disulfide bonds are formed between amino acid residues in the hinge region between said CH1 (or VLs) and CH2 domains.

In some cases, the CH2 domain of the fusion peptide is located between the scFv fragment and the CH3 domain. In other words, said scFv fragment is ligated to the CH2 terminus of said Fc fragment. In some cases the single chain unit does not contain a CH1 domain.

In one aspect, one or both of the monovalent unit and the single chain unit comprise human antibody sequences or humanized sequences. For example, in one aspect the heavy chain of the monovalent unit comprises a human or humanized Fc fragment. In one specific aspect, the heavy chain Fc fragment comprises a human IgG Fc fragment.

In one aspect, the Fc fragment of said fusion peptide comprises a human or humanized Fc fragment. In one specific aspect, the Fc fragment of said fusion peptide comprises a human IgG Fc fragment.

FIG. 1A is a schematic diagram of the structure of multifunctional antibody 1, and FIG. 1B is a schematic diagram of the primary protein structure of each component of the antibody.

Modified humanized anti-CD3 antibody of the present invention (1) CDR and FR sequences of variable region of humanized anti-CD3 antibody

(2) Sequence of novel humanized anti-CD3 antibody (partial example)

Note: Any pairing can be made between VHs and VLs.

Sequence information of antibodies (1) Antibodies targeting immune cell antigens

(2) Antibodies targeting tumor antigens or other antigens

Sequences of Other Domains (1) Amino Acid Sequence of Linker Domain

(2) Amino acid sequence of hinge domain

(3) Amino acid sequence of light chain constant region CL domain

(4) heavy chain constant region CH1 domain amino acid sequence

Fc amino acid numbering is according to Kabat numbering. "Kabat number" refers to the numbering system described by Kabat et al., the contents of which are described in the US Department of Health and Public Services, "Sequence of Proteins of Immunological Interest" (1983). Specific numbers are shown in the table below.

here,
amino acids 221-227 are the hinge domain,
amino acids 228-340 are the CH2 domain of the second constant region of the heavy chain;
Amino acids 341-447 are the CH3 domain of the heavy chain third constant region.

Antibodies may be modified to improve the efficiency of heterodimer pairing. For example, in some cases, the Fc fragment of the monovalent unit heavy chain and/or the Fc fragment of the fusion peptide has one or more substitutions (of these substitutions) compared to a wild-type antibody fragment. forming a protrusion-void structure pair between them). Protrusion-void structures are known in the art. See, for example, Ridgway et al., "'Knob-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization," Protein Engineering 9(7):617-21 (1996).

In one embodiment, T366 of one CH3 domain is replaced with a relatively large amino acid residue such as tyrosine (Y) or tryptophan (W). And Y407 of other CH3 domains may be substituted with a relatively small amino acid residue such as threonine (T), alanine (A) or valine (V).

In one aspect, one of said CH3 domains comprises one or more substitutions and is substituted with a positively charged amino acid residue under physiological conditions, and the other CH3 domain comprises one or more substitutions. , and is substituted with one or more amino acid residues that are negatively charged under physiological conditions. In one aspect, the positively charged amino acid residue may be arginine (R), histidine (H) or lysine (K). On the other hand, the negatively charged amino acid residue may be aspartic acid (D) or glutamic acid (E). Amino acid residues that may be substituted include, but are not limited to D356, L368, K392, D399 and K409.

In one embodiment, one CH3 domain has a cysteine substitution at S354 and the other CH3 domain has a cysteine substitution at Y349, with residues at both substitution positions forming a disulfide bond.

In one aspect, one CH3 domain had H435 and Y436 substituted with arginine and phenylalanine, respectively, which resulted in significantly reduced Fc and Protein A binding capacity. As a result, heterodimers and homodimers have different protein A binding activities, and can be easily separated by affinity chromatography.

Specific sequence of antigen

Example 1: Humanized Modified SP34
(1) SP34 Sequence Analysis The amino acid sequence of the SP34 heavy chain variable region (SP34VH) is shown below. Among them, the CDR region is shown in bold and underlined, and the rest is the FR region.

The amino acid sequence of the SP34 light chain variable region (SP34VL) is shown below. The CDR regions are shown in bold and underlined, the rest are FR regions.

(2) Humanization Modifications (2.1) Heavy Chain Variable Region Modifications:
All commercially available fully human or humanized antibody sequences were analyzed and the following human-derived heavy chain variable region FR sequences were selected.
(i) FR of the first group (VHFR-1), . . . means CDR.

Alignment of SP34VH with VHFR-1. Frames indicate CDR regions, - indicates regions with identical amino acids, and * indicates that there are differences in amino acids at the sites.

First Group Humanized Antibody VHs Sequences Obtained by Homology Analysis and Conservative Substitution of Amino Acids

(ii) the second group of FRs (VHFR-2), . . . means the CDRs;

Alignment of SP34VH with VHFR-2. Frames indicate CDR regions, - indicates regions with identical amino acids, and * indicates that there are differences in amino acids at the sites.

Second Group Humanized Antibody VHs Sequences Obtained by Homology Analysis and Conservative Substitution of Amino Acids

(2.2) Modification of light chain variable region:
The following human-derived FR sequences were selected:
(i) analysis of the light chain variable region sequences of all commercially available fully human or humanized antibodies yielding a first group FR (VLFR-1); ... means CDR.

Alignment of SP34VL and VLFR-1, CDR regions in frames, - indicates regions with identical amino acids, * means that there is a difference in amino acids at the site.

First Group Humanized Antibody VLs Sequences Obtained by Homology Analysis and Conservative Substitution of Amino Acids

(ii) NCBI-IgBlast search for antibody light chain variable regions with high homology to FRs of SP34VL yielding FRs of the second group (VLFR-2), . . . means CDRs.

Alignment of SP34VL and VLFR-2, CDR regions in frames, - indicates regions with identical amino acids, * means that there are differences in amino acids at the site.

Homology analysis and conservative amino acid substitutions yielded the following second group of humanized antibody VLs sequences.

Example 2: Humanized antibody preparation and antibody activity assay 1 . A method for constructing an antibody expression plasmid. The vector used is pcDNA 3.1.
(1) Target fragment DNA was amplified by polymerase chain reaction (PCR). Polymerases include DNA polymerase (2×PrimeSTAR Max Premix, TaKaRa, Item No. R405A). The DNA sequence was obtained by reverse translation from the amino acid sequences of SEQ ID NOs:45-73. The resulting PCR product is the target fragment DNA.
(2) Vector plasmids were enzymatically digested with restriction endonucleases. Restriction endonucleases include, for example, NotI, NruI or BamHI-HF. The resulting enzymatic digestion product is the enzymatically cleaved vector DNA.
* Vectors are divided into two types: heavy chain expression vector and light chain expression vector. Here, the heavy chain expression vector has the signal peptide and the DNA sequence of the constant region of the human IGG1 heavy chain, and consists of CH1, hinge, CH2 and CH3, and the heavy chain variable region. Here, the enzymatic digestion site is located between the 3' end of the signal peptide and the 5' end of CH1. The light chain expression vector contains the DNA sequences of the signal peptide and constant region of the human kappa light chain, and the light chain variable region, wherein the enzymatic digestion sites are located at the 3' end of the signal peptide and the light chain constant region. It is located between the 5' end of the region.
(3) PCR products or enzymatic digestion products were purified using Tiangen's DNA purification kit. For specific work steps, refer to the specification attached to the Tiangen kit. The purified products obtained are purified target fragment DNA and purified enzyme-digested vector DNA.
(4) genetic recombination of the target fragment; A recombinase (ExnaseII, Vazyme, Item No. C112-01) was used.
Heavy chain fragments were recombined with enzymatically digested heavy chain expression vector DNA and light chain fragments were recombined with enzymatically digested light chain expression vector DNA.
(5) Transformation by heat shock 10 μl of the recombinant product was added to 100 μl of Trans10 competent cells, mixed gently, left in an ice bath for 30 minutes, and the tube was immersed in a 42° C. water bath and held for 60 seconds. , without rocking, was quickly transferred to an ice bath and held for 2 minutes. 600 μl of LB liquid medium (containing antibiotics) was added thereto and cultured at 37° C. for 1 hour on a rotary shaker. An appropriate amount of the bacterial solution was applied onto an LB tablet containing the corresponding antibiotic, the tablet was inverted, and cultured overnight in a constant temperature incubator at 37°C. A single colony was picked and sent as a sample to Wuhan generate Biological Engineering Co., Ltd. for sequencing. A single colony with correct sequencer results is selected, expanded, and the plasmid is extracted in bulk and used for mammalian cell transfection experiments. Recombinant plasmids of heavy chain fragments and enzymatically digested heavy chain expression vector DNA are referred to as heavy chain expression plasmids. A recombinant plasmid of the light chain fragment and the enzymatically digested light chain expression vector DNA is called a light chain expression plasmid.

1. Antibody Expression Methods Two transient transfection expression systems were used here, CHO-S and 293E. Details are as follows.
(1) CHO-S transient transfection step (for example, if the total transfection volume is 100 ml)
a) The day before transfection, the cells were passaged (where eg CD-CHO can be used), the suspension cell density was adjusted to 1×10 ⁶ cells/ml and the volume was brought to 90 ml. This ensures that the cells are in logarithmic growth phase and by the time of transfection on the second day the cells can reach a density of 2×10 ⁶ cells/ml.
b) Incubated on a rotary shaker overnight at 37°C, 125 rpm, 5% CO2.
c) On the day of transfection, the FectoPRO transfection reagent was prewarmed to room temperature and mixed gently.
d) Dilute 50 μg DNA in 10 ml serum-free medium (e.g. opti PRO-SFM), mix gently, add to 100 μl transfection reagent, mix evenly and incubate at room temperature for 10 minutes to transfect. injection complexes were formed.
e) Added to 90 ml of log-phase cells that had been prepared to homogenize the transfection complex. Immediately after the addition, the mixture was shaken to homogeneity and cultured on a rotary shaker at 37°C, 125 rpm, 5% CO2.
f) 2-4 hours after transfection, 75 μl of transfection enhancer Fecto PRO® Booster was added.
g) 18-24 hours after transfection, the temperature was lowered to 32°C and cultured.
h) Supplementation of material on days 3, 5, 7 after transfection, the volume of supplementation of material is 3.5% of the total volume of cells.
i) Harvested when cell viability fell below 70%. The expression time is 9-13 days.
(2) 293E transient transfection step (for example, if the total transfection volume is 20 ml)
a) The day before transfection, pass the cells (where eg FreeStyle ^™ 293 can be used) and adjust the suspension cell density to 0.6-0.8×10 ⁶ cells/ml and bring the volume to 20 ml. made it This ensures that the cells are in logarithmic growth phase and by the time of transfection on day 2 the cells can reach a density of 1.2-1.6×10 ⁶ cells/ml. .
b) Incubated on a rotary shaker overnight at 37°C, 125 rpm, 5% CO2.
c) On the day of transfection, the LPEI was prewarmed to room temperature and mixed gently before use.
d) Dilute 20 μg DNA in 0.67 ml serum-free medium (eg FreeStyle ^™ 293) and mix well.
e) Dilute 40 μg LPEI in 0.67 ml serum-free medium (e.g. FreeStyle ^™ 293) and mix well;
f) LPEI diluted in step 5 was added to DNA diluted in step 4, mixed rapidly and incubated at room temperature for 15 minutes to form complexes.
g) To homogenize the transfection complexes from step 6, added to the prepared 20 ml of exponentially growing cells. Immediately after the addition, the mixture was shaken to homogeneity and cultured on a rotary shaker at 37°C, 125 rpm, 5% CO2.
h) Supplemented material 1 day, 3 days after transfection, the volume of material supplemented is 5% of the total cell volume.
i) Harvested antibodies expressed by day 6;
(3) The transfection was a co-transfection, in which either light chain expression plasmid and either heavy chain expression plasmid were transfected into the mammalian cells in equal proportions. The antibody obtained by expression is a monoclonal antibody and has a Y-shaped structure with bivalent symmetry consistent with natural antibodies. A schematic representation of the primary structure of this structure is shown in FIG.
(4) The abbreviations of the expressed monoclonal antibodies and their expression levels (in 293E cells) are as follows.

The specific sequences of the same heavy chain constant region and light chain constant region of the B8-B57 monoclonal antibody are as follows.

FIG. 3 shows the expression levels of humanized CD3 monoclonal antibodies. As can be seen from the antibody expression levels, monoclonal antibodies B25-B33, B35-B42, B46-B53, B56-B57, etc. have expression levels of over 15 mg/L for transient transfection.

2. Antibody Purification Affinity chromatography has been mainly used for antibody purification. Specifically:
(1) Harvesting: Antibody-expressing cell culture was centrifuged at 3000×g for 10 minutes, the supernatant was taken, filtered through a 0.22 μm filter, and stored at 4° C. waiting for use. .
(2) Affinity chromatography (MabSelect SuRe GE 17-5438-01, for example when the column volume is 18 ml)
a) Equilibration: Equilibrate column with binding buffer (25 mM Tris/trimethylolaminomethane, pH 7.0-7.4) until UV detector, conductivity reading stabilizes or remains at baseline. Equilibrate and equilibrate at least 5 column volumes.
b) Loading: The filtered supernatant was loaded at a flow rate of 5 ml/min.
c) Washing and equilibration: Flush 5 column volumes with binding buffer.
d) Elution: Samples were eluted with elution buffer (50 mM Citrate citrate, pH 3.4±0.1). Five column volumes were eluted at a flow rate of 5 ml/min and the eluted peak was collected.
e) Neutralization: Neutralize the eluate with 1M Tris trimethylolaminomethane (pH 8.0) and adjust the sample to pH 6.0±0.1. The antibody obtained by purification is a monoclonal antibody and has a Y-shaped structure with bivalent symmetry consistent with natural antibodies.
f)

3. Antibody Activity Assay In this example, the antibody activity assay is mainly an assay of binding activity of antibodies to CD3-positive cells.
1) Cell preparation: CD3-positive induced cultured CIK cells/T cells isolated from human whole blood were used for the CD3 affinity test. A sufficient amount of cells was centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS to adjust the density to 4×10 ⁶ /ml. Cells were plated at 2×10 ⁵ cells per well, adding 50 μl per well. Centrifugation was performed at 4° C. and 300×g for 5 minutes in a centrifuge, and the supernatant was discarded. Cell seeding was performed on ice.
2) Antibody addition: Antibodies were serially diluted according to the experimental design. In addition, antibody dilution was performed on ice. For example, the starting concentration of the antibody dilution was 3000 nM and was diluted to 11 gradients with 3-fold dilutions. 50 μl of the diluted antibody was added to each well containing cells, mixed well by gentle pipetting, and incubated at 4° C. for 2 hours with shaking at 1100 rpm/min.
3) Washing: Cells were resuspended in 150 μl of 1% FBS-PBS, centrifuged at 4° C. and 300×g for 5 minutes, and the supernatant was discarded. One wash was repeated.
4) Second antibody incubation: Add diluted second antibody PE anti-human IgG FC (Biolegend, 409304) to a final concentration of the second antibody of 8 µg/ml and a volume of 50 µl/well. rice field. In addition, as a control, wells containing only the cells and the second antibody were prepared, mixed well by gentle pipetting, and incubated at 4°C in the dark with shaking at 1100 rpm/min for 1 hour.
5) Washing: Cells were resuspended in 150 μl of 1% FBS-PBS, centrifuged at 4° C. and 300×g for 5 minutes, and the supernatant was discarded. One wash was repeated.
6) Fixation: 200 μl of 2% paraformaldehyde was added per well to resuspend the cells, and the cells were fixed for 20 min at room temperature. It was centrifuged at 300×g for 5 minutes and the supernatant was discarded.
7) Cell resuspension: Cells were resuspended in 200 μl 1% FBS-PBS, centrifuged at 300 g for 5 minutes, and the supernatant discarded.
8) Loading on flow cytometer: Cells were resuspended in 150 μl 1% FBS-PBS and set on flow cytometer for testing.
9) Data analysis: Data were analyzed with FlowJo 7.6, which is flow cytometer analysis software, and EC50 values were plotted and calculated with Graphpad Prism 5.

FIG. 4 shows the detection results of the binding activity of the antibodies to human and monkey T cells. FIG. 4 shows the binding ability of monoclonal antibodies to CD3 + T cells.

As can be seen from the results of detection of binding activity to cells, these antibodies all have higher affinities (EC50<100 nM) than monoclonal antibody sp34, and furthermore, to both human and monkey CD3. can also be combined.

Example 3: Preparation of multifunctional antibody of the present invention 1. Method for constructing plasmid The working steps are the same as in "1. Method for constructing antibody expression plasmid" in Example 2 of the present application. Specifically, it relates to the construction of three plasmids, a light chain expression plasmid (pL), a heavy chain expression plasmid (pH) and a fusion peptide expression plasmid (pF1).

The method for expressing the multifunctional antibody is the same as "2. Method for expressing antibody" in Example 2 of the present application. During transfection, the three plasmids were co-transfected. To express multifunctional antibody 1 shown in FIG. 1, plasmids pL, pH and pF1 together need to be transfected into CHO-S or 293E cells for expression.

The multifunctional antibody of the present invention consists of the following three polypeptides.
(1) Fusion peptide: consisting of heavy chain variable region (VHs), linker 1, light chain variable region (VLs), hinge 1, heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3-b). The sequence of Linker 1 is shown in Table 10, the sequence of Hinge 1 is shown in Table 11 for Hin2-9, the sequence for CH2 is shown in Table 19, and the sequence for CH3 is shown in Table 20 for CH3-b. The VHs and VLs sequences are all derived from the novel humanized SP34 sequence of the present application, and the details are shown in Table 2.
(2) Heavy chain: consisting of heavy chain variable region (VHm), heavy chain constant region 1 (CH1), hinge (Hinge), heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3-a). The CH1 sequence is shown in Table 13 and the hinge sequence is shown in Table 11, Hin1 sequence. The CH2 sequence is similar to the CH2 of the fusion peptide (shown in Table 19) and the CH3 sequence is shown in Table 20 as the CH3-a sequence. CH3-a and CH3-b of the fusion peptide are in one-to-one correspondence as shown in Table 20.
(3) Light chain: Consists of a light chain variable region (VLm) and a light chain constant region (CL). The sequence of CL is shown in Table 12.
A schematic diagram of the structure of the multifunctional antibody is shown in FIG. 1B.

2. Purification method of multifunctional antibody:
Antibody purification mainly used affinity chromatography, ion exchange chromatography, hydrophobic chromatography and molecular sieves. Specifically:
(1) Harvesting: Antibody-expressing cell culture was centrifuged at 3000×g for 10 minutes, the supernatant was taken, filtered through a 0.22 μm filter, and stored at 4° C. waiting for use. .
(2) Affinity chromatography (MabSelect SuRe GE 17-5438-01, for example when the column volume is 18 ml)
a) Equilibration: Equilibrate the column with binding buffer (25 mM trimethylolaminomethane, pH 7.0-7.4) until the UV detector, conductivity reading stabilizes or remains at baseline, and at least Five column volumes were equilibrated.
b) Loading: The filtered supernatant was loaded at a flow rate of 5 ml/min.
c) Washing and equilibration: Flush 5 column volumes with binding buffer.
d) Elution: Samples were eluted with elution buffer (50 mM citric acid, pH 3.4±0.1). Five column volumes were eluted at a flow rate of 5 ml/min and the eluted peak was collected.
e) Neutralization: Neutralize the eluate with 1M Tris/trimethylolaminomethane (pH 8.0) and adjust the sample to pH 6.0±0.1.
(3) ion-exchange layering (using cation-exchange chromatography as an example, HiTrap SP-HP GE 17-1151-01, 5 ml column volume);
a) Preparation of sample: After microfiltration of the sample for affinity chromatography, it was diluted with ultrapure water so that the conductivity was 5 mS/cm or less. The pH was then adjusted to 6.0±0.1.
b) Equilibration and loading: First, 5 column volumes of buffer B (25 mM citric acid + 1 M sodium chloride, conductivity should be 80-90 mS/cm, pH should be 6.0 ± 0.1 ). In addition, the conductivity, pH, UV-based The column was equilibrated until the line stabilized and then loaded at a flow rate of 3 ml/min.
c) Washing/Equilibration: The column was washed with 5 times the column volume of buffer A at a flow rate of 5 ml/min.
d) Elution: eluted with 0-30% buffer B, 20 column volumes, eluted with 100% buffer B, 10 column volumes. The flow rate was 3 ml/min during the whole process. The eluate was collected and detected in different tubes.
(4) Hydrophobic chromatography (Capto phenyl ImpRes, filler: GE XK16/20 11.5 cm/23 ml)
a) Sample treatment: with 5M sodium chloride, the sample was adjusted to 1M sodium chloride and the pH was adjusted to 6.0.
b) Equilibration and loading: First equilibrate with buffer A (25 mM Citrate citrate + 1 M sodium chloride, pH 6.0 ± 0.1) with 5 column volumes at a flow rate of 5 ml/min. turned into Then, it was loaded at a flow rate of 3.3 ml/min.
c) Washing/Equilibration: The column was washed with 5 times the column volume of buffer A at a flow rate of 5 ml/min. Washed with 10% buffer B (25 mM Citrate citrate, pH is 6.0±0.1) using 5 column volumes at a flow rate of 5 ml/min.
d) Elution: Elution was performed with 90% buffer B at a flow rate of 5 ml/min, and elution peaks were collected and detected in different tubes.
(5) Molecular sieves (HiLoad Superdex 200pg GE 28989336 26/600)
a) Equilibration and loading: After equilibration with buffer (20 mM histidine + 0.15 M sodium chloride, pH 6.0 ± 0.1) using 2 column volumes, loading was carried out at a flow rate of 3 ml/min.
Elution: Eluted with buffer twice the column volume and collected the elution peak in different tubes for detection.

Abbreviations for some of the specific expressed antibodies and the corresponding antibody variable region amino acid sequences are provided in the table below.

Expression levels of the multifunctional antibodies of the invention are shown in FIG. Figure 5 shows expression levels of transient transfections of multifunctional antibodies assembled from humanized anti-CD3 antibodies in CHO cells.

As can be seen from FIG. 5, in CHO cells, expression levels of transient transfection showed some differences in multifunctional antibodies assembled from different anti-CD3 antibodies. In Y102, Y150-F8-3/F8-4/F8-6/F8-7/F8-8/F8-9/F8-10/F9-11/F9-12, MS-hCD3-IC15/IC16/IC17 , the expression level is significantly higher, and the antibody expression level is 40 mg/L or higher.

Example 4 Biological Activity Assay of Multifunctional Antibodies 1. Affinity with Cells 1) Cell preparation: Using T cells that are CD3 positive isolated from human whole blood, CD3 A terminal affinity test was performed and a tumor antigen affinity test was performed using tumor cells positive for the corresponding antigen. For example, in the CD38 antigen test, CD38-positive MM.1S cells (purchased from Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences) or RPMI 8226 cells (purchased from Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences) are used. , the PD-L1 antigen test uses PD-L1-positive H358 cells (purchased from Cell Resource Center, Shanghai Institute of Life Science, Chinese Academy of Sciences). A sufficient amount of cells was centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS and adjusted to a density of 4×10 ⁶ /ml. Cells were plated at 2×10 ⁵ cells per well, adding 50 μl per well. After centrifugation at 300×g at 4° C. for 5 min, the supernatant was discarded. Cell seeding was performed on ice.
2) Antibody addition: Antibodies were serially diluted according to the experimental design. In addition, antibody dilution was performed on ice. For example, the initial concentration of the antibody dilution was 3000 nM and was diluted to 11 gradients with 3-fold dilutions. 50 μl of the diluted antibody was added to each well containing cells, mixed well by gentle pipetting, and incubated at 4° C. for 2 hours with shaking at 1100 rpm/min.
3) Washing: Cells were resuspended in 150 μl of 1% FBS-PBS, centrifuged at 4° C. and 300×g for 5 minutes, and the supernatant was discarded. One wash was repeated.
4) Second antibody incubation: second antibody PE anti-human IgG FC (Biolegend, 409304) diluted so that the final concentration of the second antibody is 8 ug/ml and the volume is 50 μl/well was added. In addition, as a control, wells containing only the cells and the second antibody were prepared, mixed well by gentle pipetting, and incubated at 4°C in the dark with shaking at 1100 rpm/min for 1 hour.
5) Washing: Cells were resuspended in 150 μl of 1% FBS-PBS, centrifuged at 4° C. and 300 g for 5 minutes, and the supernatant was discarded. One wash was repeated.
6) Fixation: 200 μl of 2% paraformaldehyde was added per well to resuspend the cells, and the cells were fixed for 20 min at room temperature. It was centrifuged at 300×g for 5 minutes and the supernatant was discarded.
7) Cell resuspension: Cells were resuspended in 200 μl 1% FBS-PBS, centrifuged at 300×g for 5 min, and the supernatant was discarded;
8) Loading to flow cytometer: Cells were resuspended in 150 μl 1% FBS-PBS, set in flow cytometer for detection.
9) Data analysis: Data were analyzed with FlowJo 7.6, which is flow cytometer analysis software, and Kd values were plotted and calculated with Graphpad Prism 5.

2. Activation of T cells 1) Using well-cultured tumor cells (non-small cell lung cancer cells H358, purchased from Cell Resource Center, Shanghai Institute of Life Science, Chinese Academy of Sciences; myeloma cells MC/CAR, purchased from ATCC), Single cell suspensions were prepared and seeded in 96-well plates at a cell number of 2E4/well.
2) PBMCs were isolated from whole blood of healthy volunteers by density gradient centrifugation. PBMCs were added to tumor cell-seeded plates according to the experimental design E:Tratio (effector cells: target cells).
3) According to the experimental design, the antibody was diluted in a series of concentration steps and each concentration of antibody was added.
4) The 96-well plate was incubated in a 37°C, 5% CO2 incubator until the test time. Suspended PBMC cells were pooled and the corresponding CD3, CD69 test antibodies were added, incubated for 1 h, and excess antibody was washed away. The cells were resuspended and the percentage of CD3 and CD69 double-positive cells was detected by flow cytometer, which was taken as the activated percentage of T cells in the antibody-induced PBMC. Specifically, it was calculated by the following formula.
Percentage of CD3 and CD69 double positive cells (%) = number of CD3 and CD69 double positive cells/total number of CD3 positive cells x 100%
Using "GraphPad Prism 5" software, non-linear fitting (log (agonist) vs. response--Variable slope) is performed with the biobody concentration on the horizontal axis and the CD3 & CD69) % value on the vertical axis to calculate the T cell activation curve and EC50 value. bottom.

3. In Vitro Cytotoxicity 1) A sufficient amount of tumor cells (eg H358 cells and MC/CAR cells) was used to prepare a single cell suspension.
2) CFSE staining of tumor cells: A certain amount of cell suspension was centrifuged (300×g, 5 min) to remove the supernatant. A CFSE solution prepared in 2 ml PBS was added so that the final concentration of CFSE was 5 μM. Cells were placed in a 5% CO2, 37°C incubator and incubated for 15 min. Cells were removed from the incubator, washed with PBS, centrifuged (300 xg, 5 min) and the supernatant removed. The washing was repeated three times, the cells were resuspended in complete medium, the suspension taken and counted.
3) Seeding of tumor cells: Cells were resuspended in complete medium to a cell density of 2 x ¹⁰⁵ /ml and added to a 96-well plate at 2 x ¹⁰⁴ cells/well, ie 100 µl/well.
4) Addition of effector cells, PBMC (isolated from human whole blood): resuspend the effector cells in the complete medium used for the tumor cells in the experimental system, and add the corresponding effector cells calculated according to the E:Tratio of the experimental design. amount was added to the plate. Addition volume is 50 μl/well.
5) Addition of diluted antibody: According to the experimental design, the highest concentration of antibody is 10 μg/ml. If 50 μl of antibody is added, it corresponds to 1/4 of the total volume of 200 μl, so each should be prepared to 4 times the above concentration before adding the antibody. Briefly, antibodies were initially diluted to 40 μg/ml and starting at 40 μg/ml with 10-fold dilutions (9 steps, volume 50 μl/well).
6) The 96-well plate was observed under a microscope to confirm that the cells were uniformly dispersed in the culture wells, and then placed in a cell incubator at 37°C in 5% CO2 and cultured, waiting for detection.
7) After reaching the detection time, the treatment for adherent cells was: Aspirate the cell supernatant, wash with PBS at 30 μl/well, aspirate the wash and combine with the previous aspirated supernatant. Add 30 μl/well of trypsin into the cell wells and place in a cell incubator at 37° C., 5% CO 2 for 3-5 min digestion, add the previously collected supernatant, and sieve each well until a single cell suspension. Cells were pipetted. Treatment for suspension cells: Mixed evenly by pipetting multiple times.
8) PI (10 μg/ml final concentration) was added to each sample at 10 ul/well 10-15 min prior to loading on the flow cytometer.
9) Flow cytometer detection; Flow cytometer detection results were analyzed with FlowJo software, data analysis was exported with Microsoft Excel, and a form was created with GraphPad for analysis. Calculation formula for lethality: The ratio of CFSE- and PI-double positive cells to CFSE-positive cells was defined as the death rate of the target cells.
The calculation formula is as follows.
Target cell mortality (%) = number of pI and CFSE double-positive cells/number of CFES-positive cells x 100
10) Calculation of the ability of the antibody to kill tumor cells: Calculate the target cell mortality at all antibody concentrations according to the target cell mortality calculation formula, and plot the antibody concentration as the horizontal axis and the target cell mortality as the vertical axis. created a curve. Graphpad Prism 5 was used as data analysis software, and the obtained EC50 value was defined as the cell-killing ability of the antibody.

Specific binding activities of some of the multifunctional antibodies are shown in Table 28.

Specific T cell activation abilities of some of the multifunctional antibodies are shown in FIG. 7 and Table 29.

The cell-killing ability of some of the multifunctional antibodies is shown in FIGS. 8 and 9 and Tables 30 and 31.

Comparative Example 1:
1. Sequence alignment of humanized antibody and existing anti-CD3 antibody:
The variable region sequences of anti-CD3 antibody 1 are described in patent US8846042B2, in which the heavy chain variable region corresponds to SEQ ID NO: 44 of said patent and the light chain variable region corresponds to SEQ ID NO: 56 of said patent. .

The variable region sequences of anti-CD3 antibody 2 are described in patent US9650446B2, in which the heavy chain variable region corresponds to SEQ ID NO: 85 of said patent and the light chain variable region corresponds to SEQ ID NO: 194 of said patent. .

(1) Alignment of heavy chain variable regions:

The amino acid sequence similarity between VH2a and anti-CD3 antibody 1 VH was 96.8%, and differences were observed between FR-H1 and FR-H4.

The amino acid sequence similarity between VH2a and anti-CD3 antibody 2 VH was 95.2%, and differences were observed in FR-H1, FR-H2 and CDR-H2.

(2) Alignment of light chain variable regions:

The amino acid sequence similarity between VL5 and anti-CD3 antibody 1 VL was 94.5%, and differences were observed in FR-L1, FR-L3 and FR-L4.

The amino acid sequence similarity between VL5 and anti-CD3 antibody 2 VL was 96.3%, and differences were observed in FR-L1, CDR-L1, FR-L3 and FR-L4.

2. Comparison of biological activities between humanized antibodies and existing anti-CD3 antibodies, monoclonal antibodies:
(1) Affinity test of existing anti-CD3 antibodies Table 26 Abbreviations and amino acid sequences of existing anti-CD3 antibodies

The method for producing an existing monoclonal antibody was the same as the antibody production method in Examples 2 and 3. Data from the cell-binding activity assay are shown in FIG. FIG. 6 shows the affinity of existing anti-CD3 antibodies, monoclonal antibodies, to cells.

Comparative Example 2:
(1) Partial Abbreviations and Variable Region Amino Acid Sequences of Comparative Multifunctional Antibodies Specific examples are shown in the table below.

(2) Cell affinity test of the multifunctional antibody of the present invention and comparative multifunctional antibody The results of the affinity test of the multifunctional antibody are shown in the table below.

From Table 28, it was observed that when the CD3 humanized antibody of the present invention is combined with different CD38 monoclonal antibodies to form a multifunctional antibody, the affinities of both ends are affected to different extents.

(3) Testing the activation level of the multifunctional antibody of the present invention for T cells The results of testing the activation level of MS-hCD3-IC-17 on human T cells are shown in FIG. 5:1, processing time: 48 h). FIG. 7 showed the ability of different multifunctional antibodies of the invention to activate T cells in vitro.

A “*” indicated that the platform stage had not been reached and the curve fitting was inaccurate. Actual EC50 values may be higher than those shown in the table.

In FIG. 7, Y150-F8-9 has the strongest ability to activate T cells, and both the antibody and the ability to bind to two antigens (CD38, CD3) are among the antibodies in the figure. Strongest. Y150-F8-8 has a level of T cell activation ability similar to that of Y150-F8-9 (data not shown). Antibodies Y150-F8-9, Y150-F8-10 and Y150-F8-12 have perfectly matched anti-CD38 antibody sequences and also have a perfectly matched affinity for CD38, but these antibodies are have anti-CD3 antibody sequences that are not different, differ in affinity for CD3 by 1-3 amino acid point mutations, and also have significant differences in their ability to activate T cells. Y150-F8-9 has the strongest ability to activate T cells, followed by Y150-F8-10, and Y150-F8-12 has weak ability to activate T cells. Y150-F9-11 has significant T-cell activation function.

(4) Test of cell-killing ability of multifunctional antibody of the present invention and comparative multifunctional antibody Figure 8 shows the test results of the ability of 1 to kill multiple myeloma cells MC/CAR (here, the ratio of the numbers of effector cells, human PBMC, and target cells, MC/CAR, was 5:1). and processing time: 72 h):

Figure 8 showed the in vitro killing ability of different multifunctional antibodies against multiple myeloma cells MC/CAR.

In FIG. 8, Y150-8-5 and Y150-8-6 show the other portions of the antibody (including Fab and Fc) except that the anti-CD3 scFv sequences were both novel humanized anti-CD3 antibody sequences. The sequence was completely identical to Y150-F8-1. The anti-CD3 scFv sequence of Y150-F8-1 was that of the known antibody SP34. As can be seen from the above data, the multifunctional antibodies of the present invention have similar or better tumor cell killing ability. Since the control antibody is non-killing, the cell-killing ability of the multifunctional antibody is generated by bispecific targeted binding to tumor or immune cells to induce immune cell-to-tumor cell attack. It was shown that A CD38 monoclonal antibody is the commercially available CD38 antibody drug DARZALEX®.

The results of a test of the ability of Y105, a multifunctional antibody of the present invention, and Y106, a comparative multifunctional antibody, to kill lung cancer cell H358 are shown in FIG. The number ratio of MC/CAR was 10:1, treatment time: 48 h).

Figure 9 showed the In Vitro killing ability of different multifunctional antibodies against lung cancer cell H358.

In Figure 9, Y105 was completely identical to Y106 in the sequence of the rest of the antibody (including Fab and Fc), except that the anti-CD3 scFv sequence was the novel humanized anti-CD3 antibody sequence. The anti-CD3 scFv sequence of Y106 is that of the known antibody SP34. As can be seen from the above data, the multifunctional antibodies of the present invention have similar or better tumor cell killing ability. Given the weak killing of control antibodies, the cytotoxicity of multifunctional antibodies is generated by bispecific targeting and binding to tumor or immune cells to induce immune cell-to-tumor cell attack. It was shown that The S70 mAb was the marketed PD-L1 antibody drug Tencentriq®.

(5) Stability test of the multifunctional antibody of the present invention and comparative multifunctional antibody Experiment 1: Thermal acceleration stability test at 40 ^° C. The specific work steps were as follows.
1, the sample is replaced with a specific buffer having the following composition: (a) citrate buffer: 20 mM citric acid, pH 5.5, or (b) histidine buffer: 50 mM histidine, pH 5.5, and Concentration was adjusted to 1 mg/mL.
2. 500 μL of sample was placed in each tube, sealed, and left in a water bath at 40 ^° C. Samples were taken every 24 hours for HPLC-SEC detection, and the water bathing time was 14 days in total.

The detection results are shown in Figures 10-16.

FIG. 10 shows an accelerated thermal stability test at 40 ^° C. in a citrate buffer system for multifunctional antibody Y105 of the invention, comparative antibodies Y106, CT-F4, CT-F5 and CT-F6. As can be seen from Figure 10, the multifunctional antibodies of the invention have good thermostability. The antibody did not change significantly after 14 days of 40°C treatment, which was similar to the comparative antibody Y100 and significantly superior to CT-F4, CT-F5 and CT-F6. Here, Y105 has the same Fab and Fc sequences as all comparator antibodies, but a different ScFv. Y105 has the anti-CD3 antibody VH2a and VL5 sequences of the invention. Y106 is the SP34 antibody sequence, CT-F4 is the anti-CD3 antibody 1 sequence, CT-F5 is the anti-CD3 antibody 2 sequence, and CT-F6 has the anti-CD3 antibody 1 VH and anti-CD3 antibody 2 VL sequences.

FIG. 11 shows the multifunctional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7, Y150-F8-8 of the present invention, comparative antibodies Y150-F8-1, Y150-F8-2, CT- An accelerated thermal stability test at 40 ^° C. in a citrate buffer system was shown for F1, CT-F2 and CT-F3. All multifunctional antibodies in Figure 11 have the same Fab sequence, Y150-F8-5, Y150-F8-6, Y150-F8-1, CT-F1, the same Fc has an array. The anti-CD3 antibody sequences of Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 are VH2a and VL5, and the anti-CD3 of Y150-F8-1 and Y150-F8-2. The antibody sequence is SP34, the anti-CD3 antibody sequence of CT-F1 is anti-CD3 antibody 1, the anti-CD3 antibody sequence of CT-F2 is anti-CD3 antibody 2, and the anti-CD3 antibody sequence of CT-F3 were anti-CD3 antibody 1 VH and anti-CD3 antibody 2 VL. As can be seen from the data in the figure, the multifunctional antibodies of the invention have significantly better thermal stability than the comparative antibodies.

FIG. 12 shows the multifunctional antibodies Y150-F8-9, F8-10, F8-12, F9-7, F9-11 of the invention, comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, An accelerated thermal stability test at 40 ^° C. in a citrate buffer system was shown for CT-F2 and CT-F3. Y150-F8-9, F8-10, F8-12, F9-7, and F9-11 in FIG. 12 are multifunctional antibodies of the present invention, and their anti-CD3 antibody sequences are VH2a and VL5, or VH2j and VL5a (F8-10), or VH21 and VL5b (F8-12). Y150-F8-1, Y150-F9-6, CT-F1, CT-F2, CT-F3 are comparative multifunctional antibodies whose CD3 sequences are different from SP34 (Y150-F8-1, Y150-F9- 6), anti-CD3 antibody 1 (CT-F1), anti-CD3 antibody 2 (CT-F2), anti-CD3 antibody 1VH and anti-CD3 antibody 2VL (CT-F3). As can be seen from the data in Figure 12, the multifunctional antibodies of the invention have significantly better thermal stability than the comparative antibodies.

Figure 13 shows accelerated thermal stability studies at 40 ^° C in citrate buffer system for multifunctional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 of the invention and comparative antibodies IC-2 to IC-7. . All antibodies in FIG. 13 had a perfect match in Fab sequence. Among them, MS-hCD3-IC15, IC16, IC17 and IC18 were multifunctional antibodies of the present invention, and their anti-CD3 antibody sequences were VH2a and VL5 or VH2j and VL5a (IC18). IC-2 through IC-7 are comparative multifunctional antibodies, and their anti-CD3 antibody sequences are SP34 (IC-2 and IC-3), anti-CD3 antibody 1 (IC-4), anti-CD3 antibody 2, respectively. (IC-5), anti-CD3 antibody 1VH and anti-CD3 antibody 2VL (IC-6 and IC-7). As can be seen from the data in the figure, the multifunctional antibodies of the invention have significantly better thermal stability than the comparative antibodies.

FIG. 14 shows multifunctional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7, Y150-F8-8 of the present invention, comparative antibodies Y150-F8-1, Y150-F8-2, CT- An accelerated thermal stability test at 40 ^° C. in histidine buffer system was shown for F1, CT-F2 and CT-F3. All multifunctional antibodies in FIG. 14 have the same Fab sequence, Y150-F8-5, Y150-F8-6, Y150-F8-1, CT-F1, the same It has an Fc sequence. The anti-CD3 antibody sequences of Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 are VH2a and VL5, and the anti-CD3 antibodies of Y150-F8-1 and Y150-F8-2 The sequence is SP34, the anti-CD3 antibody sequence of CT-F1 is anti-CD3 antibody 1, the anti-CD3 antibody sequence of CT-F2 is anti-CD3 antibody 2, and the anti-CD3 antibody sequence of CT-F3 is anti-CD3 antibody 1 VH and 2 VL of anti-CD3 antibody. As can be seen from the data in the figure, all multifunctional antibodies of the invention have good thermostability. Comparative antibodies Y150-F8-1, Y150-F8-2, CT-F1, CT-F2, CT-F3 have significantly weaker thermal stability than the multifunctional antibodies of the invention.

FIG. 15 shows multifunctional antibodies Y150-F8-9, F8-10, F8-12, F9-7, F9-11 of the invention, comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, An accelerated thermal stability test at 40 ^° C. in a histidine buffer system was shown for CT-F2 and CT-F3. In FIG. 15, Y150-F8-9, F8-10, F8-12, F9-7, and F9-11 are multifunctional antibodies of the present invention, and their anti-CD3 antibody sequences are VH2a and VL5, or VH2j. VL5a (F8-10), or VH21 and VL5b (F8-12). Y150-F8-1, Y150-F9-6, CT-F1, CT-F2, CT-F3 are comparative multifunctional antibodies whose CD3 sequences are identical to SP34 (Y150-F8-1 and Y150-F9- 6), anti-CD3 antibody 1 (CT-F1), anti-CD3 antibody 2 (CT-F2), anti-CD3 antibody 1VH and anti-CD3 antibody 2VL (CT-F3). As can be seen from the data in Figure 15, the multifunctional antibodies of the invention have significantly better thermal stability than the comparative antibodies.

FIG. 16 shows accelerated thermal stability studies at 40 ^° C. in histidine buffer system for multifunctional antibodies MS-hCD3-IC15, IC16, IC17, IC18 of the invention and comparative antibodies IC-2 through IC-7. rice field. The Fab sequences of all antibodies in Figure 16 were a perfect match. Among them, MS-hCD3-IC15, IC16, IC17 and IC18 were multifunctional antibodies of the present invention, and their anti-CD3 antibody sequences were VH2a and VL5 or VH2j and VL5a (IC18). IC-2 through IC-7 are comparative multifunctional antibodies, and their anti-CD3 antibody sequences are SP34 (IC-2 and IC-3), anti-CD3 antibody 1 (IC-4), anti-CD3 antibody 2, respectively. (IC-5), anti-CD3 antibody 1VH and anti-CD3 antibody 2VL (IC-6 and IC-7). As can be seen from the data in the figure, the multifunctional antibodies of the invention have significantly better thermal stability than the comparative antibodies.

Experiment 2: Stability test at low pH.
Stability at low pH, also called acid tolerance, investigates whether antibody molecules can maintain their original state when treated in an acidic environment for a period of time and then neutralized to physiological conditions again. is an indicator. Specifically, in the protein A affinity chromatography of antibody molecules, the antibody solution eluted (using a citrate buffer of pH 3.5) in the acid elution step is not neutralized, but in the buffer After maintaining for a certain period of time, samples were taken at 30 minutes and 60 minutes, neutralized by adding 1/10 volume of 1 M Tris-HCl (pH 8.0), and the samples were subjected to HPLC-SEC test. rice field.

FIG. 17 shows acid resistance stability test in pH 3.5 citrate buffer system for multifunctional antibody Y105 of the present invention, comparative antibody Y106, CT-F4, CT-F5 and CT-F6. As can be seen from Figure 17, the multifunctional antibody of the present invention has good acid resistance. The antibody showed no significant change even after 60 min of treatment at low pH, and had significantly better acid resistance than Y106, CT-F4, CT-F5 and CT-F6.

FIG. 18 shows multifunctional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7, Y150-F8-8 of the present invention, comparative antibodies Y150-F8-1, Y150-F8-2, CT- For F1, CT-F2, and CT-F3, an acid resistance stability test in a pH 3.5 citrate buffer system was shown. All multifunctional antibodies in FIG. 18 have the same Fab sequence, Y150-F8-5, Y150-F8-6, Y150-F8-1, CT-F1, the same It has an Fc sequence. The anti-CD3 antibody sequences of Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 are VH2a and VL5, and the anti-CD3 of Y150-F8-1 and Y150-F8-2. The antibody sequence is SP34, the anti-CD3 antibody sequence of CT-F1 is anti-CD3 antibody 1, the anti-CD3 antibody sequence of CT-F2 is anti-CD3 antibody 2, and the anti-CD3 antibody sequence of CT-F3 were anti-CD3 antibody 1 VH and anti-CD3 antibody 2 VL. As can be seen from the data in the figure, the multifunctional antibodies of the invention have significantly better acid resistance than the comparative antibodies.

Figure 19 Multifunctional antibodies Y150-F8-9, F8-10, F8-12, F9-7, F9-11 of the present invention, comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT- For F2 and CT-F3, an acid resistance stability test in a pH 3.5 citrate buffer system was shown. Y150-F8-9, Y150-F9-7, and Y150-F9-11 in FIG. 19 are multifunctional antibodies of the present invention, and their anti-CD3 antibody sequences are VH2a and VL5, or VH2j and VL5a (F8-10 ), or VH21 and VL5b (F8-12). Y150-F8-1, CT-F1, CT-F2, CT-F3 are comparative multifunctional antibodies whose CD3 sequences are similar to SP34 (Y150-F8-1 and Y150-F9-6), anti-CD3 antibodies, respectively. 1 (CT-F1), anti-CD3 antibody 2 (CT-F2), anti-CD3 antibody 1VH and anti-CD3 antibody 2VL (CT-F3). As can be seen from the data in Figure 19, the multifunctional antibodies of the invention have significantly better acid resistance than the comparative antibodies.

Figure 20 shows the acid resistance test in a pH 3.5 citrate buffer system for the multifunctional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 of the invention and the comparative antibodies IC-2 to IC-7. rice field. The Fab sequences of all antibodies in Figure 20 were a perfect match. Among them, MS-hCD3-IC15, IC16, IC17 and IC18 were multifunctional antibodies of the present invention, and their anti-CD3 antibody sequences were VH2a and VL5 or VH2j and VL5a (IC18). IC-2 through IC-7 are comparative multifunctional antibodies, and their anti-CD3 antibody sequences are SP34 (IC-2 and IC-3), anti-CD3 antibody 1 (IC-4), anti-CD3 antibody, respectively. 2 (IC-5), anti-CD3 antibody 1VH and anti-CD3 antibody 2VL (IC-6 and IC-7). As can be seen from the data in the figure, the multifunctional antibodies of the invention have significantly better acid resistance than the comparative antibodies.

(6) In vivo efficacy test of the multifunctional antibody of the present invention and a comparative multifunctional antibody 1, test material:
Cells: Daudi (human multiple myeloma cell line, purchased from ATCC), human PBMC;
Mice: NOD/SCID, 5 weeks old, female, Beijing vital River laboratory animal technology Co., Ltd. , Ltd. .

Inoculation method: Daudi cells were inoculated subcutaneously on the right back, human PBMC were inoculated from the tail vein at D0, and both Daudi and PBMC cells were inoculated.

Study drugs: (B) Y150-F8-8, (C) Y150-F8-9, (D) Y150-F9-11.
Negative controls: (A) blank control; (H) MS-hCD3-IC-17.
Positive control: (G) CD38 mAb (CD38 monoclonal antibody, Darzalex®).

Administration method: (1) Y150-F8-8, Y150-F8-9, Y150-F9-11 and MS-hCD3-17 were designed to have different doses and were administered via the tail vein, and administration was D0. It started with TIW (3 times a week) x 2. (2) CD38 mAb was administered on D0 and D7, with a dose of 5 mg/kg for D0 and 15 mg/kg for D7. There were 6 animals in each group.

Body weight: Body weight was measured three times a week during the dosing period, and then twice a week thereafter.

Tumor volume: Tumor latency period was 9-20 days, and after the average tumor volume reached 30 mm3, tumor length and width were measured twice a week. All remaining tumor-bearing mice were photographed for about 30 days or when the mean tumor volume in the negative control group approached 2000 mm 3 as the monitoring time. Also, for each group, the group was terminated when the tumor volume of an entire group approached 2000 mm3 or when the tumor volume of one mouse reached 3000 mm3.

2. Experimental Results The experimental results of Y150-F8-8 are shown in FIG. 21A, the experimental results of Y150-F8-9 are shown in FIG. 21B, and the experimental results of Y150-F9-11 are shown in FIG. 21C.

Figure 21 showed the in vivo effects of different multifunctional antibodies and tumor volume monitoring in a mouse tumor model. Among them, Y150-F8-8 (A), F8-9 (B) and F9-11 (C) are all multifunctional antibodies of the present invention, and their anti-CD3 antibody sequences are VH2a and VL5. and each had a different anti-CD38 antibody sequence.

From FIG. 21, it can be seen that the multifunctional antibodies Y150-F8-8, F8-9 and F9-11 of the present invention have a significant tumor suppressive effect, with no significant difference compared to the control monoclonal antibody. It was also found that the effective dosage could completely suppress the tumor, and animals did not show any significant toxic reactions or side effects.

(7) Toxicity test of the multifunctional antibody of the present invention in monkeys 2F5 mAb is an anti-CD38 monoclonal antibody and can cross-reactively bind to human monkey CD38. Its specific array is:

Multifunctional antibodies Y150-F8-10, F9-11, F9-12, comparative antibody Y150-F9-6 and monoclonal antibody 2F5 mAb were each subjected to toxicity tests in monkeys. A single intravenous infusion was administered, and the doses are shown in the table below.

In the Y150-F8-10, F9-11, F9-12 groups, the numbers of the monkey lymphocyte subset CD38 + CD20 + cells were significantly reduced until 24 h after administration, reaching complete elimination. In the 2F5mAb group (dosage: 20 mg/kg), the number of cells in this group was significantly reduced to about 30% of that before administration, and was not completely eliminated. From these data, Y150-F8-10, F9-11, F9-12 molecules were found to have significant CD38+ cell elimination effect, and as can be seen from Table 34, Y150-F8-10, Y150- F9-11 and Y150-F9-12 have weaker toxicity than Y150-F9-6.

Claims (21)

A humanized antibody or antigen-binding fragment thereof,
specifically binds to CD3 of humans and/or primates such as monkeys;
comprising framework regions FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3 and FR-L4, and complementarity determining regions (CDRs);
The amino acid sequences of CDR1 and CDR2 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NOs: 1 and 2, respectively, and the amino acid sequence of CDR3 of the heavy chain variable region is SEQ ID NO: 3 or a variant sequence thereof. any one of the sequences shown in 4, 10, 11, 12, 13, 14 or 191;
the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28, respectively;
The framework region of the humanized antibody is
a) FR-H1 of SEQ ID NO: 16
b) FR-H2 of SEQ ID NO: 17
c) FR-H3 of SEQ ID NO: 23 or any one of its variant sequences SEQ ID NOS: 19, 20 and 24
d) FR-H4 of SEQ ID NO:25
e) FR-L1 of SEQ ID NO:31
f) FR-L2 of SEQ ID NO: 36 or one of its variant sequences SEQ ID NO: 33, 34
g) FR-L3 of SEQ ID NO:41, and h) FR-L4 of SEQ ID NO:43
A humanized antibody or antigen-binding fragment thereof comprising the sequence of comprising a heavy chain variable region and a light chain variable region,
The heavy chain variable region is
a) the amino acid sequence of SEQ ID NO: 49 or 58, or b) the amino acid sequence of SEQ ID NO: 49 or 58 and 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, The light chain variable region comprises any one sequence selected from the group of amino acid sequences having 97%, 98%, 99% or more amino acid sequence identity,
d) the amino acid sequence of SEQ ID NO: 71, 72 or 73, or e) the amino acid sequence of SEQ ID NO: 71, 72 or 73 and 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , any one sequence selected from the group of amino acid sequences having 96%, 97%, 98%, 99% or more amino acid sequence identity,
Preferably, the heavy chain variable region and the light chain variable region are each
SEQ ID NO: 49, 71; SEQ ID NO: 49, 73; SEQ ID NO: 58, 72
2. The humanized antibody or antigen-binding fragment thereof of claim 1, comprising an amino acid sequence selected from the group of a multispecific antibody, preferably a bispecific antibody,
comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and an antibody or antigen-binding fragment against another antigen and / or other antigenic epitope,
proteins whose other antigens and/or other antigenic epitopes are e.g. overexpressed on tumor cells compared to corresponding non-tumor cells; tumor antigens e.g. CD38, BCMA, PD-L1, SLAMF7, Claudin 18.2 or CEA; virus; bacteria; and/or endotoxin;
multispecific antibodies. an antibody,
(a) a light chain-heavy chain pair and (b) a fusion peptide, wherein said light chain-heavy chain pair has specificity for tumor cells or microorganisms, and said fusion peptide comprises a single chain variable region. comprising a fragment and a single chain Fc fragment, having specificity for immune cells, wherein said single chain variable region fragment comprises a heavy chain variable region of the amino acid sequence of any one of claims 1-2 and An antibody comprising a light chain variable region. said Fc fragment comprises CH2 having a sequence of any one of SEQ ID NOS: 155-161 or 192, and/or CH3 having a sequence of any one of SEQ ID NOS: 162-183, and/or said fusion peptide is VHs-linker1-VLs-hinge1-CH2-CH3-b, wherein said heavy chain comprises VHm-CH1-hinge2-CH2-CH3-a and said light chain comprises VLm-CL;
5. The antibody of claim 4. said light chain-heavy chain pair is a) a protein overexpressed on tumor cells compared to corresponding non-tumor cells;
b) tumor antigens such as CD38, BCMA, PD-L1, SLAMF7, Claudin 18.2 or CEA,
c) viruses;
d) bacteria, and/or e) endotoxins,
The antibody of any one of claims 4-5, which specifically binds to. a substitution in which said heavy chain, the heavy chain of said fusion peptide and/or the Fc fragment of said fusion peptide forms a protrusion-cavity pairing between said heavy chain and said fusion peptide compared to a wild-type antibody , including one or more of the substitutions of, for example, Table 15;
For example, T366 of one CH3 domain is replaced with a relatively large amino acid residue such as tyrosine (Y) or tryptophan (W), and Y407 of the other CH3 domain is replaced with a relatively small amino acid residue such as threonine (T ), alanine (A) or valine (V), and/or the Fc fragment of said heavy chain and/or said fusion peptide comprises one or more substitutions,
1) said substitution is a substitution that forms a salt bridge pairing between said heavy chain and said fusion peptide, e.g. comprising one or more substitutions of Table 16;
For example, one CH3 domain contains one or more substitutions of amino acid residues that are positively charged under physiological conditions, and the other CH3 domain contains one or more substitutions of amino acid residues that are negatively charged under physiological conditions. including
The positively charged amino acid residue is, for example, arginine (R), histidine (H), or lysine (K), and the negatively charged amino acid residue is, for example, aspartic acid (D) or glutamic acid (E). optionally, the substituted amino acid residue is, for example, one or more of D356, L368, K392, D399 and K409;
2) said substitution is a substitution that forms a disulfide bond between said heavy chain and said fusion peptide, e.g., a substitution in Table 17; and/or
3) said substitution is a substitution that resulted in a significant reduction in binding ability to Fc and Protein A, e.g., H435 and Y436 of one CH3 domain are replaced with arginine and phenylalanine, respectively;
The antibody according to any one of claims 4-6. VH of said fusion peptide comprises the sequence of SEQ ID NO: 49 or 58;
the VL of the fusion peptide comprises the sequence of SEQ ID NO: 71, 72 or 73;
linker 1 of the fusion peptide comprises the sequence of any one of SEQ ID NOS: 120-138;
hinge 1 of the fusion peptide and hinge 2 of the heavy chain comprise the sequence of any one of SEQ ID NOS: 139-147;
CH2 of the fusion peptide, CH2 of the heavy chain comprises the sequence of any one of SEQ ID NOs: 155-161 or 192;
CH3-b of said fusion peptide comprises the sequence of any one of SEQ ID NOs: 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183;
CH3-a of said heavy chain comprises the sequence of any one of SEQ ID NOs: 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182;
VHm of said heavy chain comprises the sequence of any one of SEQ ID NOs: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 193;
CH1 of said heavy chain comprises the sequence of SEQ ID NO: 154;
the light chain VLm comprises the sequence of any one of SEQ ID NOs: 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 194; and / or
6. The antibody of claim 5, wherein the CL of said light chain comprises the sequence of any one of SEQ ID NOs: 148-153. VH of the fusion peptide and VL of the fusion peptide each comprise an amino acid sequence selected from the following group,
a) SEQ ID NO: 49, 71; SEQ ID NO: 49, 73; SEQ ID NO: 58, 72
b) at least one amino acid sequence of a) and 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or wherein VHm of said heavy chain and VLm of said light chain each comprise an amino acid sequence selected from the group of:
d) SEQ ID NOs: 90, 91; SEQ ID NOs: 92, 93; SEQ ID NOs: 94, 95; SEQ ID NOs: 96, 97; 108, 109; 110, 111; 112, 113; 114, 115; 116, 117; 118, 119; ) at least one amino acid sequence of d) and 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of An amino acid sequence with amino acid sequence identity. a) CH3-b of said fusion peptide and CH3-a of said heavy chain have a substitution pair that forms a protrusion-cavity structure;
b) CH3-b of said fusion peptide and CH3-a of said heavy chain have a substitution pair that forms an ionic bond;
c) CH3-b of said fusion peptide and CH3-a of said heavy chain have a substitution pair that forms a disulfide bond, and/or d) CH3-b of said fusion peptide and CH3-a of said heavy chain has a substitution that resulted in reduced ability to bind to protein A,
6. The antibody of claim 5. Y150-F9-12, Y105, Y150-F8-5, Y150-F8-7, Y150-F8-8, Y150-F8-9, Y150-F8-10, Y150-F9-7, Y150-F9-11, selected from MS-hCD3-IC15, MS-hCD3-IC16, MS-hCD3-IC17 and MS-hCD3-IC18;
Here, each component follows the order of fusion peptide VHs-linker1-VLs-hinge1-CH2-CH3-b, heavy chain VHm-CH1-hinge2-CH2-CH3-a, light chain VLm-CL,
Y150-F9-12 is SEQ ID NO: 49, 129, 71, 144, 192, 167, 92, 154, 139, 192, 166, 93, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
Y105 is 80%, 85%, 90% with SEQ ID NOs: 49, 129, 71, 142, 139, 167, 106, 154, 139, 159, 166, 107, 148, respectively, or at least one of these amino acid sequences , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
Y150-F8-5 is SEQ ID NO: 49, 129, 71, 141, 139, 167, 90, 154, 139, 157, 166, 91, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
Y150-F8-7 is SEQ ID NO: 49, 129, 71, 144, 158, 167, 90, 154, 139, 158, 166, 91, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
Y150-F8-8 is SEQ ID NO: 49, 129, 71, 144, 161, 167, 90, 154, 139, 161, 166, 91, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
Y150-F8-9 is SEQ ID NO: 49, 129, 71, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
Y150-F8-10 is SEQ ID NO: 58, 129, 72, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
Y150-F9-7 is SEQ ID NO: 49, 129, 71, 141, 139, 167, 92, 154, 139, 157, 166, 93, 150, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
Y150-F9-11 is SEQ ID NO: 49, 129, 71, 144, 161, 167, 92, 154, 139, 161, 166, 93, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
MS-hCD3-IC15 is SEQ ID NO: 49, 129, 71, 141, 159, 167, 118, 154, 139, 159, 166, 119, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
MS-hCD3-IC16 is SEQ ID NO: 49, 129, 71, 141, 157, 167, 118, 154, 139, 157, 166, 119, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
MS-hCD3-IC17 is SEQ ID NO: 49, 129, 71, 141, 161, 167, 118, 154, 139, 161, 166, 119, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
MS-hCD3-IC18 is SEQ ID NO: 58, 129, 72, 141, 161, 167, 118, 154, 139, 161, 166, 119, 148, respectively, or at least one of these amino acid sequences and 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity,
The antibody according to any one of claims 4-10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 4 to 11,
binds to a target with a KD of about ^10-8 M or less, such as about ^10-8 M, ^10-9 M, ^10-10 M or less, or about 100 nM or less, such as about 10 nM, 1 nM, 0. target with an EC50 of 9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less Preferably, said antigen-binding fragment is a fusion peptide in the antibody of any one of claims 4-11 or an F(ab) derived from the antibody of any one of claims 1-3. ') an antibody or antigen-binding fragment thereof selected from 2, Fab', Fab, Fv, Fd, scFv. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-3 and 4-12. An expression vector comprising the polynucleotide of claim 13 . A host cell comprising the polynucleotide of claim 13 or the expression vector of claim 14. A method for preparing the antibody according to any one of claims 1 to 3 and 4 to 12,
15. A method comprising preparing said antibody by introducing the polynucleotide of claim 13 or the expression vector of claim 14 into a host cell. an antibody conjugate,
comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 4 to 12 and a conjugate portion conjugated thereto,
Preferably, the antibody conjugate, wherein said conjugate moiety is selected from purification tags (e.g. His tags), cytotoxic agents, detectable labels, radioisotopes, luminescent substances, colored substances, enzymes or polyethylene glycol. . A fusion protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3 and 4-12. the antibody or antigen-binding fragment thereof of any one of claims 1-3 and 4-12, the antibody conjugate of claim 17, or the fusion protein of claim 18,
optionally further comprising a pharmaceutically acceptable carrier and/or excipient,
Preferably a dosage form suitable for oral administration to the gastrointestinal (GI) tract, preferably said dosage form is a tablet, capsule, pill, powder, granule, emulsion, microemulsion, solution, in a dosage form selected from suspensions, syrups and elixirs; or in a dosage form suitable for subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.
pharmaceutical composition. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3 and 4-12, the antibody conjugate of claim 17, or the fusion protein of claim 18, , preferably, the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-3 and 4-12, the antibody conjugate according to claim 17, or the antibody according to claim 18. A kit further comprising a second antibody that specifically recognizes the fusion protein; optionally, said second antibody further comprising a detectable label such as a radioisotope, luminescent substance, colored substance or enzyme. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 4 to 12 for the treatment of diseases,
Preferably, the disease comprises a cancer or tumor, such as multiple myeloma, lung cancer such as small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc. Antibody or antigen-binding fragment thereof .